A Schizochytrium strain capable of efficiently converting food waste into single-cell protein and its application

Abstract

The present invention discloses a Schizochytrium strain that efficiently converts food waste to produce single-cell protein and its application, belonging to the field of bioengineering technology. The Schizochytrium spLOX‑2 deposit number is: CGMCC No.41623. The present invention also discloses a method for efficiently converting food waste to produce single-cell protein, fermenting food waste to produce volatile fatty acids; and using it as a carbon source to culture Schizochytrium to obtain single-cell protein. The recombinant strain spLOX‑2 in the present invention has improved utilization of lactic acid, and its biomass and protein yields are increased by 14.19% and 12% respectively compared with the wild-type strain, and the oil content is significantly increased by 107.1%. The present invention provides a new path for a sustainable bioeconomic model, in which waste is converted into valuable biochemicals, laying a solid foundation for expanding the scale of the process and the economic feasibility in industrial applications.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and more particularly to a Schizochytrium strain capable of efficiently converting food waste into single-cell protein and its application. Background Art

[0002] Faced with the global protein shortage, biomanufacturing technology is considered one of the key solutions. Biomanufacturing utilizes microorganisms, enzymes, or cell factories to synthesize target products through biotransformation, demonstrating tremendous potential in the food, pharmaceutical, and chemical industries. In recent years, microbial-based protein production, such as single-cell protein (SCP), has been recognized as an effective alternative to traditional protein sources such as animal and plant proteins. However, traditional biomanufacturing still faces a key bottleneck: the availability of low-cost raw materials. Currently, most biomanufacturing processes rely on expensive sugars, starches, or other fermentable organic matter as feedstock, which not only increases production costs but also intensifies competition for food resources. Furthermore, traditional microbial cultivation typically requires complex pretreatment processes, resulting in high process costs, making it difficult to remain competitive in industrial applications. Therefore, finding inexpensive, efficient, and sustainable sources of raw materials is a key breakthrough in the field of biomanufacturing.

[0003] Food waste is a potential source of raw materials for biomanufacturing, offering the advantages of abundant resources and low cost. Its main components include carbohydrates, proteins, fats, and other organic substances, and possess high biodegradability. However, due to its complex composition, its direct utilization presents numerous technical challenges, such as bacterial contamination and low conversion efficiency. Therefore, the efficient conversion of food waste into usable raw materials for biomanufacturing remains a key research challenge.

[0004] In recent years, the rapid development of synthetic biology has provided a new technical path for the high-value utilization of food waste. Synthetic biology is used to modify microorganisms so that they can efficiently convert specific substrates and produce target biological products. These products are widely used in various industries such as food, cosmetics, chemicals and textiles, which can achieve the reuse and value-added of waste. The "circular bioeconomic model" of microbial-based waste biorefineries aims to recycle and reuse waste streams by combining the biological transformation stage with microbial growth to produce high-value products and renewable biofuels. However, due to low biomass yields, high downstream processing costs and other capital requirements, microbial cell-based factories cannot produce biochemicals that are both economical and sustainable. Therefore, in the microbial fermentation process, only the co-production of high biomass and multiple value-added chemicals can achieve the maximum value-added benefits of waste streams. Summary of the Invention

[0005] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0006] Another object of the present invention is to provide a Schizochytrium strain that can efficiently convert food waste into single-cell protein and its application.

[0007] To this end, the technical solution provided by the present invention is:

[0008] A Schizochytrium strain that efficiently converts food waste into single-cell protein, the Schizochytrium strain is Schizochytrium spLOX-2, and the classification name of the Schizochytrium spLOX-2 strain is: Schizochytrium Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. spLOX-2 is deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) with the deposit number of CGMCC No. 41623 and the deposit date of November 11, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The application of the Schizochytrium strain in the treatment of kitchen waste and the production of single-cell protein.

[0010] A method for efficiently converting food waste into single-cell protein comprises the following steps:

[0011] 1) Fermenting food waste to produce volatile fatty acids (VFAs);

[0012] 2) Cultivating Schizochytrium using the volatile fatty acids produced in step 1) as a carbon source, and obtaining single-cell protein from the cultured cells after several days of cultivation. More preferably, in step 2), the culture is carried out for 6 days.

[0013] Preferably, in the method for efficiently converting food waste to produce single-cell protein, in step 1), fermenting food waste to produce volatile fatty acids comprises the following steps:

[0014] The food waste is dried and made into food waste powder. Then, the food waste powder, the original inoculum seed liquid and water are placed in a sealed container, and the air is removed to form an anaerobic environment. Then, oxygen is injected into the sealed container and anaerobic digestion is carried out under micro-aeration conditions for 1 to 8 days to obtain a fermented product of the food waste and harvest the fermentation liquid.

[0015] Preferably, in the method for efficiently converting food waste into single-cell protein, in step 1), during the anaerobic digestion, the initial OL of the fermentation product is 5-25 g VS / L, i.e., each liter of fermentation product contains 5 to 25 grams of food waste volatile solids (VS), and an oxygen injection rate of 6 mL / g VS (i.e., 6 mL of oxygen is injected per gram of VS) (typically measured at standard temperature and pressure). More preferably, the fermentation temperature is 37°C.

[0016] Preferably, the method for efficiently converting food waste into single-cell protein further comprises the following steps:

[0017] The lactate oxidase LOX gene from Aeromonas viridans is codon-optimized, or the lactate dehydrogenase d-LDH gene and l-LDH gene from Schizochytrium are transformed into Schizochytrium to obtain three recombinant Schizochytrium strains. In step 2), the Schizochytrium uses any one or more of the three recombinant Schizochytrium strains.

[0018] Preferably, in the method for efficiently converting food waste to produce single-cell protein, in step 2), the Schizochytrium spLOX-2 is Schizochytrium spLOX-2, and the classification name of the Schizochytrium spLOX-2 strain is: Schizochytrium spLOX-2 Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. spLOX-2 is deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) with the deposit number of CGMCC No. 41623 and the deposit date of November 11, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0019] Preferably, in the method for efficiently converting food waste to produce single-cell protein, in step 2), the volatile fatty acids produced in step 1) are used as a carbon source by adding a fermentation medium for Schizochytrium to the fermentation broth of the food waste fermentation in step 1), adjusting the initial pH to 6-9 to form a final fermentation medium, and culturing Schizochytrium in the final fermentation medium.

[0020] Preferably, in the method for efficiently converting food waste to produce single-cell protein, the fermentation medium for Schizochytrium contains the following components at concentrations: 5 g / L yeast extract, 0.3 g / L NaCl, 1 g / L K2SO4, 0.1 g / L KH2PO4, 4 g / L MgSO4·7H2O, and 0.05 g / L CaCl2.

[0021] Preferably, in the method for efficiently converting food waste to produce single-cell protein, in step 1), the ratio of food waste powder to original inoculum seed liquid is 1:10 (v / v).

[0022] Preferably, in the method for efficiently converting food waste into single-cell protein, the Schizochytrium is Schizochytrium SR21. More preferably, the culture temperature is 28°C, and the initial pH is adjusted to 6-7.

[0023] The present invention has at least the following beneficial effects:

[0024] The increasing generation of municipal solid waste (MSW), particularly food waste (FW), poses severe global environmental challenges and resource management challenges. This study utilizes micro-aeration / thermal pretreatment to enhance the biodegradability of FW, converting it into valuable volatile fatty acids (VFAs). Schizochytrium then utilizes the VFAs to produce lipid-rich single-cell protein, thereby achieving value-added utilization of FW. Results showed that under oxygenated conditions with an organic loading rate of 20 gVS / L, VFA production significantly increased to 37.0 g / L, demonstrating the effectiveness of oxygen treatment in increasing VFA production. Furthermore, the present invention discovered that Schizochytrium SR21 has excellent VFA utilization capacity. This discovery opens new avenues for microbial utilization of FW. Experiments demonstrated that at a pH of 7 and a VFA content of 100%, the strain exhibited strong VFA consumption, with short-chain VFA concentrations decreasing significantly over time, demonstrating its efficient metabolic process. Furthermore, by expressing exogenous lactate oxidase, the lactic acid consumption capacity was significantly improved, and the biomass, oil content and protein yield of the recombinant strain increased significantly, reaching a maximum of 17.46 g / L, 14.87% and 6.53 g / L. Among them, the biomass and protein yield increased by 14.19% and 12% respectively compared with the wild type, while the oil content increased significantly by 107.1%. The present invention provides a new path for a sustainable bioeconomic model and proposes a sustainable and efficient protein production path, aiming to solve the global protein shortage problem and promote the resource utilization of food waste. In this model, waste is converted into valuable biochemicals, which not only conforms to the concept of circular economy and green manufacturing, but also provides a new research direction and practical basis for the sustainable development of the future food industry.

[0025] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the design of the experimental device in the present invention.

[0027] Figure 2This is a graph showing changes in lactic acid concentration under different organic loads with and without anaerobic pretreatment conditions in the present invention.

[0028] Figure 3 This is a graph showing changes in acetic acid concentration under different organic load conditions with and without anaerobic pretreatment in the present invention.

[0029] Figure 4 This is a graph showing the change in butyrate concentration under different organic load conditions with and without anaerobic pretreatment in the present invention.

[0030] Figure 5 This is a graph showing the change in formic acid concentration under different organic load conditions with and without oxygen pretreatment in the present invention.

[0031] Figure 6 This is a graph showing changes in propionic acid concentration under different organic load conditions with and without anaerobic pretreatment in the present invention.

[0032] Figure 7 This is a graph showing the changes in total VFA concentration under different organic loading conditions with or without oxygen pretreatment in the present invention.

[0033] Figure 8 This is a graph showing changes in ammonium nitrogen concentration under different organic loads with and without anaerobic pretreatment conditions in the present invention.

[0034] Figure 9 This is a graph showing changes in chemical oxygen demand (COD) concentration under different organic loads with and without anaerobic pretreatment conditions in the present invention.

[0035] Figure 10 Under different initial pH conditions in the present invention Schizochytriumlimacinum The consumption of VFA by SR21 during fermentation is shown in Figure 2. (a) shows the consumption of VFA by Schizochytrium at an initial pH of 6; (b) shows the consumption of VFA by Schizochytrium at an initial pH of 7; (c) shows the consumption of VFA by Schizochytrium at an initial pH of 8; and (d) shows the consumption of VFA by Schizochytrium at an initial pH of 9.

[0036] Figure 11 Under different initial pH conditions in the present invention Schizochytriumlimacinum Fermentation parameter diagram of SR21 using VFA fermentation to produce single-cell protein. a is the pH change of the fermentation liquid during the VFA culture of Schizochytrium under different initial pH conditions; b is the biomass, protein content, and protein yield detected after the completion of VFA fermentation of Schizochytrium under different initial pH conditions; c is the oil content and fatty acid composition and content detected after the completion of VFA fermentation of Schizochytrium under the initial pH conditions.

[0037] Figure 12 Under the conditions of different concentrations of VFA in the present invention Schizochytriumlimacinum The figure shows the consumption of VFA by Schizochytrium under the condition of 25% VFA; b shows the consumption of VFA by Schizochytrium under the condition of 50% VFA; c shows the consumption of VFA by Schizochytrium under the condition of 75% VFA; and d shows the consumption of VFA by Schizochytrium under the condition of 100% VFA.

[0038] Figure 13 Under the conditions of different concentrations of VFA in the present invention Schizochytriumlimacinum Fermentation parameter diagram of SR21 using VFA fermentation to produce single-cell protein. a is the pH change of the fermentation liquid during the VFA culture of Schizochytrium under different VFA concentrations; b is the biomass, protein content, and protein yield detected after the VFA fermentation of Schizochytrium under different VFA concentrations; c is the oil content and fatty acid composition and content detected after the VFA fermentation of Schizochytrium under different VFA concentrations.

[0039] Figure 14 Figure 2 is a partial metabolic pathway diagram of Schizochytrium in the present invention.

[0040] Figure 15 Figures 1 and 2 show the plasmid construction and recombinant strain verification used in the present invention for Schizochytrium. a) shows the plasmid construction for overexpressing genes in Schizochytrium; b) shows the PCR verification of the recombinant strain; and c) shows the qualitative detection of the reporter gene β-glucosidase (GUS) expressed in the recombinant strain.

[0041] Figure 16 This is a diagram showing the consumption of VFAs during the fermentation culture of Schizochytrium in the present invention, wherein a is the consumption of VFAs by the recombinant strain d-LDH; b is the consumption of VFAs by the recombinant strain l-LDH; c is the consumption of VFAs by the recombinant strain spLOX; d is the consumption of VFAs by the recombinant strain PE; and e is the consumption of VFAs by the wild-type strain SR21.

[0042] Figure 17Figure 1 is a fermentation parameter diagram for the production of single-cell protein by VFA fermentation of Schizochytrium sp. of the present invention. a shows the pH change of the fermentation broth during VFA culture of the recombinant and wild-type Schizochytrium sp. strains; b shows the total VFA consumption and conversion rate detected after the completion of VFA fermentation of the recombinant and wild-type strains; c shows the biomass, protein content, and protein yield detected after the completion of VFA fermentation of the recombinant and wild-type strains; and d shows the oil content and fatty acid composition and content detected after the completion of VFA fermentation of the recombinant and wild-type strains.

[0043] The taxonomic name of the Schizochytrium spLOX-2 strain is: Schizochytrium Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. spLOX-2 is deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) with the deposit number of CGMCC No. 41623 and the deposit date of November 11, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0044] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0045] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0046] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0047] Schizochytrium is a promising industrial microorganism due to its rapid growth rate, high cell density, and tolerance to shear stress. It can metabolize a variety of organic carbon sources (such as glucose, glycerol, volatile fatty acids (VFAs), and waste fermentation broths) to produce abundant polyunsaturated fatty acids (such as DHA) and small amounts of high-value products such as squalene and astaxanthin, providing added value for its application in functional foods and feeds. However, glucose production is costly, with glucose as a carbon source accounting for up to 80% of production costs. Compared to glucose or other carbon sources, the use of VFAs can significantly reduce the cost of microbial fermentation. Furthermore, since VFAs undergo a shorter metabolic pathway than sugar substrates, their conversion and synthesis of secondary metabolites are enhanced. Therefore, utilizing Schizochytrium to process VFAs in food waste fermentation broths not only reduces production costs but also enables resource recovery.

[0048] In this invention, micro-aeration and thermal pretreatment are combined to enhance enzymatic hydrolysis, improve VFA production efficiency, and accelerate anaerobic digestion (AD) rates. This combined approach leverages the strengths of each pretreatment method to maximize the effectiveness of the overall process. Micro-aeration pretreatment involves partially exposing microorganisms to oxygen under moderate operating conditions, accelerating the hydrolysis phase by promoting microbial activity and cell growth. Thermal pretreatment is typically used because it is cost-effective and requires fewer chemicals than other methods. Studies have shown that thermal pretreatment can increase VFA yields and AD rates.

[0049] In this study, the main research objectives were: (1) to investigate the synergistic effects of combined pretreatment (including micro-aeration and thermal pretreatment) on enhancing enzymatic hydrolysis, improving VFA production efficiency, and accelerating AD rate; (2) to investigate the effect of organic load changes on VFA production during AD batch processing; and (3) to explore the feasibility of Schizochytrium utilising VFAs from food waste and, by expressing an exogenous lactate oxidase gene, to enable Schizochytrium to effectively use VFAs as substrates and produce single-cell proteins rich in high-value lipids. By combining AD with microbial fermentation, this study on FW treatment provides a new strategy for the successful development of a variety of value-added chemicals.

[0050] 1 Materials and Methods

[0051] 1.1 Raw materials and inoculum

[0052] FW was collected from the cafeteria of the Tianjin Institute of Industrial Biotechnology, China, dried in an oven, and ground using a high-speed grinder (Beijing Xingshi Lihe) before being added to the fermentation system. Biogas slurry (BS) and a liquid mixture were purchased online (Taobao, China) and used as the original inoculum for the AD process. To reduce methanogenic activity, the original inoculum was heated to 80°C for 25 minutes and then added at a 1:10 (v / v) ratio.

[0053] 1.2 Micro-aeration / thermal pretreatment and organic load (OL)

[0054] Before addition to the reactor, food waste (FW) was oven-dried at 100°C for 72 hours and ground into a powder using a high-speed grinder. The composition is shown in Table 2. After thermal pretreatment, the FW was placed in 1 L bottles for oxygen pretreatment. During pretreatment, 10 g of FW, 20 mL of the original inoculum seed solution, and deionized water were mixed to a total volume of 100 mL. The bottles were then sealed with rubber stoppers and flushed with nitrogen for 3 minutes to expel all air. After all air was expelled from the bottles, 6 mL / g VS oxygen was added to each bottle at atmospheric pressure using a syringe and labeled as O2 pretreatment. The oxygen pretreatment process was repeated, and two bottles were left unpretreated as controls (labeled as non-oxygen pretreated). During oxygen pretreatment, all bottles were incubated in a shaker at 37°C and 100 rpm for 24 hours.

[0055] This experiment employed five different organic loading (OL) rates: 5, 10, 15, 20, and 25 VSg / L. Three replicates of each OL were used, with both aerobic and anaerobic pretreatments to evaluate the effects of oxygen on various OL and VFA production. The pH was set at 7.0 at the start of the experiment, and the temperature was maintained at 37°C throughout the experiment, which lasted eight days. The optimal organic loading rate (OLR) was determined to maximize VFA production.

[0056] 1.3 Analytical methods

[0057] The total solids (TS) and volatile solids (VS) of FW and BS were determined according to previously described analytical methods (APHA, 2005). After recording the weights of FW and BS, the samples were dried at 105°C for 24 hours. After 24 hours, the FW and PS were weighed again to determine the TS content, as shown in Equation 1. In a second step, the dried samples were heated in a muffle furnace to 550°C for two hours to reduce them to ash. The samples were removed from the furnace, cooled to room temperature, and then weighed again. The VS content was calculated as shown in Equation 2.

[0058]

[0059] Among them, m1 is the weight of the empty crucible, m2 is the weight of the crucible and raw materials before drying, m3 is the weight of the crucible and raw materials after drying, and m4 is the weight of the crucible and ash after the muffle furnace.

[0060] Assuming that the sum of C, H, O, and N constitutes 99.5% of volatile solids (VS), the C, H, N, and O contents were quantified using an elemental analyzer. The C, H, N, and O contents in food waste were determined using data from VS. An automated pH meter (AquaSearcher™, OHAUS, China) was used to measure pH. COD and ammonia nitrogen were determined using HACH kits (HACH, USA) according to the kit instructions. Lactic acid determination and VFA analysis were performed using a high-performance liquid chromatography (HPLC) system (Agilent) equipped with an ICSep ICE-Coregel 87H3 column (USA). The mobile phase consisted of 5 mM H₂SO₄ solution at a flow rate of 0.6 mL / min and a pressure of 60 kPa.

[0061] 1.4 Anaerobic Digestion (AD)

[0062] Anaerobic digestion was repeated under aerobic and anaerobic pretreatment conditions. A feedstock to inoculum ratio of 1:10 (v / v) was used, and concentrated BS was used to inoculate both aerobically and anaerobically pretreated FW in a working volume of 0.8 mL. To ensure anaerobic conditions, anaerobic bottles were filled with high-purity nitrogen, sealed, and incubated in a shaker at 37°C at 130 rpm for 8 days. Samples were collected daily for analysis of VFA and lactate concentrations. Figure 1 shows a schematic diagram of the experimental setup.

[0063] 1.5 Strains, culture media, and culture conditions

[0064] E. coli strains were grown in Luria–Bertani medium containing 50 mg / L kanamycin at 37°C and 180 rpm.

[0065] The present invention uses SchizochytriumlimacinumThe SR21 strain was maintained in our laboratory as a frozen culture. Cells stored in 50% (v / v) glycerol at -80°C were cultured in two stages: a seed culture and a fermentation culture. The seed culture contained glucose (30 g / L), yeast extract (8 g / L), and artificial sea crystals (20 g / L). The fermentation culture consisted of yeast extract (5 g / L), NaCl (0.3 g / L), K₂SO₄ (1 g / L), KH₂PO₄ (0.1 g / L), MgSO₄·7H₂O (4 g / L), and CaCl₂ (0.05 g / L). The FW fermentation product was centrifuged to obtain the fermentation broth, which was then filtered to remove impurities. The fermentation medium components were added to the filtered fermentation broth, and the VFAs in the fermentation broth served as a carbon source for Schizochytrium schizochytrium cultivation at 28°C and a shaking speed of 180 rpm. VFA fermentation media with different initial pH conditions consisted of sterilized treated VFA fermentation media. The pH of the fermentation broth was then adjusted to 5, 6, 7, 8, and 9 using 5 M NaOH. The pH of the culture medium was monitored using a portable pH meter. All other culture conditions remained unchanged. The VFA concentrations of the fermentation media were determined using fermentation broths containing 25% (13.27 g / L) VFA, 50% (24.93 g / L) VFA, 75% (36.42 g / L) VFA, and 100% (44.34 g / L) VFA as the carbon source. The remaining fermentation media components were then added, stirred, and sterilized. The pH was then adjusted using 5 M NaOH. All media were sterilized at 115°C for 20 minutes.

[0066] 1.6 Plasmid construction

[0067] Escherichia coli DH5α was used for plasmid construction and plasmid enrichment. Schizochytriumlimacinum Lactate dehydrogenase (d-LDH) and l-LDH genes were predicted in the SR21 genome. The d-LDH and l-LDH genes were amplified from Schizochytrium genomic DNA using corresponding primers and inserted into the pK2-PA-(ApaI / KpnI)cyc-PE-NPTII vector digested with ApaI and KpnI. The lactate oxidase (LOX) gene from Aeromonas viridans was codon-optimized, and the amplified SpLOX fragment was similarly inserted into the pK2-PA-(ApaI / KpnI)cyc-PE-NPTII vector digested with ApaI and KpnI. PCR primer synthesis, sequencing, and gene synthesis services were provided by Suzhou Jinweizhi Biotechnology Co., Ltd. for this experiment. See Appendix Table 1 for specific primers.

[0068] Table 1. Primers used in the present invention

[0069]

[0070] 1.7 Transformation of Schizochytrium

[0071] The constructed vector was transformed into Schizochytrium using particle bombardment cell method. Schizochytrium The sp. culture was centrifuged at 6000 rpm for 5 minutes, washed twice with 20 mL of sterile water, and the waste liquid discarded. The cells were resuspended in 1 mL of sterile water, and 200 μL of the culture was spread onto a seed plate and air-dried to fix. Microparticle preparation: 5 mg of 0.6 µm Gold Microcarriers (Bio-Rad, USA), 4-6 μg of plasmid, 20 μL of 0.1 M spermidine, and 50 μL of 2.5 M CaCl₂. The mixture was then washed twice with 200 μL of anhydrous ethanol and centrifuged at 6000 rpm to obtain DNA-coated gold microparticle carriers. The gold microparticle carriers were then resuspended in 70 μL of ethanol to serve as bullets for particle bombardment. A solution containing DNA-coated gold microparticles was plated onto a microparticle plate. The microparticles were then bombarded into cells using a Biolistic PDS-1000 / He system (Bio-Rad, USA) with parameters set at 1100 psi and a target distance of 6 cm. Following bombardment, the plate was incubated at 28°C for 16–24 hours. The cells were then plated onto solid seed plates containing 150 mg / ml of LG418 and incubated at 28°C for 5–7 days to select for transformants.

[0072] Individual colonies on the resistant plates were stained using a GUS staining kit (Coolaber, SL7160) to screen for transformants. Transformants were passaged five times and further verified by PCR amplification of the target gene.

[0073] 1.8 Determination of biomass and protein content

[0074] We used the same method as previously published studies to estimate biomass using dry cell weight (DCW). After culture completion, an equal volume (V) of bacterial culture was transferred to a 50 mL pre-weighed centrifuge tube (M1) and centrifuged at 6000 rpm for 10 minutes at 4°C. The cells were washed three times with distilled water and frozen overnight in a -20°C freezer. The cells were then freeze-dried using a freeze dryer (FD-1C-50, BJBYK, China). The centrifuge tube and cells were weighed and recorded as M2. Dry cell weight was calculated using Equation 3.

[0075]

[0076] Protein content was determined using a previously published method. Freeze-dried cells were ground into a powder, and an appropriate amount of powder (3–4 mg) was weighed. Nitrogen content (%) was measured using a Flashsmart elemental analyzer (Thermo Ltd., Waltham, MA). Protein content (%) was calculated using Equation 4.

[0077]

[0078] 1.9 Lipid content determination and fatty acid composition analysis

[0079] Total lipid content was determined using a modified method from our earlier studies. Fatty acids in the lipids were converted to fatty acid methyl esters (FAMEs) using a gas chromatograph (GC-2010, Shimadzu, Japan). The gas chromatograph temperature was initially set to 180°C and then increased to 240°C at a rate of 30°C / min over 18 minutes. Temperatures of 250°C and 260°C were selected for the injection port and flame ionization port, respectively. The injection volume was 1 μL, with a split ratio of 30:1 and nitrogen as the carrier gas.

[0080] 1.10 Statistical Analysis

[0081] All data and graphs were processed using Origin Pro 9.0 (Origin Lab, USA). All data were subjected to analysis of variance (ANOVA) and multiple comparisons to determine significant differences in experimental data, and a P value of less than 0.05 was considered statistically significant.

[0082] 2 Results and Discussion

[0083] 2.1 Characteristics of food waste and inoculum

[0084] Table 2 lists the characteristics of the FW and inoculum used in this study. The TS (35.3%) and VS (10.5%) of FW were higher than those of the inoculum (TS: 1.6%, VS: 1.5%). The VS / TS ratio of FW was 29.6%, while the VS / TS ratio of the inoculum was significantly higher, at 94.6%. The initial pH of FW was 5.3, and that of the inoculum was 8.0; both were adjusted to 7.0. The nitrogen content of FW (49.3%) was higher than that of the inoculum (3.6%), while the carbon content of the inoculum was 25.8% higher than that of FW (4.0%). In addition, FW had higher hydrogen (6.8%) and oxygen (37.2%) contents than the inoculum. The C / N ratio of FW was 12.3, while that of the inoculum was 7.2, indicating that a balanced nutrient composition favored VFA production via AD in FW fermentation.

[0085] Table 2. Characteristics of food waste and inoculum

[0086]

[0087] A represents the total weight of the sample, sample, B represents TS

[0088] 2.2 VFA production with and without micro-aeration pretreatment and under different OL conditions

[0089] To better understand the degradation of FW and the production of target compounds, the present inventors conducted batch fermentation experiments, focusing on the production of VFAs from FW. The results illuminated the complex relationship between microbial interactions and metabolic processes during VFA synthesis. Specifically, the present inventors examined VFA composition and synthesis under different OL conditions (5, 10, 15, 20, and 25 gVS / L) with and without micro-aeration and thermal pretreatment, providing insight into the impact of these pretreatment techniques on the process.

[0090] 2.2.1. Lactic acid production

[0091] As shown in Figure 2, with and without micro-aeration pretreatment, lactate production significantly increased with increasing organic loading rate (OLR) from 5 to 25 gVS / L, driven by both the OLR and the presence of oxygen. At the lowest OL of 5 gVS / L, lactate production increased slightly from 2.1 g / L to 10.3 g / L in the absence of oxygen and to 11.1 g / L in the presence of oxygen over 8 days. Furthermore, at OLs of 10 and 15 gVS / L, the increase in lactate production due to oxygen pretreatment became more pronounced, reaching 17.3 and 21.3 g / L, respectively. At an OLR of 20 gVS / L, a significant increase in lactate production was observed, reaching 26.9 g / L in the presence of oxygen, compared to 22.8 g / L in the absence of oxygen. Interestingly, at the highest OL of 25 gVS / L, lactate production decreased slightly, reaching 18.9 g / L in the absence of oxygen and increasing to 19.6 g / L in the presence of oxygen. High concentrations of lactic acid tend to inhibit the activity of methanogenic microorganisms, and lactic acid is an important indicator of AD stability. Micro-aeration pretreatment consistently enhanced lactic acid production in all OLs, indicating that it plays a key role in improving fermentation efficiency and may help break down complex substrates into sugars that are more easily fermented by lactic acid bacteria (LAB). The mechanism of lactic acid production involves anaerobic fermentation of carbohydrates by LAB. Initially, carbohydrates are broken down into glucose and then converted to pyruvate through glycolysis. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase, a process that regenerates NAD from NADH. + , allowing glycolysis to proceed. This biochemical pathway not only highlights the importance of optimal conditions for LAB activity but also illustrates the key role of oxygen in potentially enhancing the initial breakdown of complex carbohydrates, thereby making the substrate more accessible for fermentation. These findings emphasize the importance of balancing OLR and oxygen pretreatment to optimize lactate production, a key parameter in industrial fermentation processes aimed at achieving efficient lactate production.

[0092] 2.2.2. Formation of acetic acid

[0093] Figure 3 shows the effects of oxygen ionization (OL) levels and the presence of oxygen on acetate production. Acetate production was extremely low at the lowest OL level of 5 gVS / L, but increased slightly in the presence of oxygen. As OL levels increased, yields under anaerobic conditions consistently decreased compared to those under aerobic conditions. This trend was evident at all OL levels, with the most significant increases observed at higher OL levels (20 gVS / L and 25 gVS / L), where anaerobic yields decreased compared to aerobic yields, reaching 3.4 g / L and 4.0 g / L, respectively, compared to 4.5 g / L and 4.4 g / L, respectively. These results clearly demonstrate that the introduction of oxygen into this anaerobic process can enhance the efficiency of AD by selectively inhibiting or promoting certain microbial activities. Oxygen can inhibit methanogenic archaea due to their oxygen sensitivity, thereby reducing their competition with acidogens for substrate. This results in increased production of volatile fatty acids, including acetate, due to increased substrate availability for acidogens. However, excess oxygen leads to fully aerobic digestion, which reduces acetic acid production as acetic acid further decomposes into carbon dioxide and water. Acetic acid production involves two main pathways: aerobic fermentation by Acetobacter, which oxidizes ethanol to acetic acid in the presence of oxygen; and anaerobic fermentation by Clostridium, which converts hexoses to acetic acid in the absence of oxygen. The presence of oxygen not only influences metabolic pathway selection but also increases overall acetic acid production. Therefore, acetic acid production requires appropriate micro-aeration.

[0094] 2.2.3. Butyrate production

[0095] Butyrate production varied with OL levels, with significant differences observed when comparing aerobic and anaerobic conditions, as shown in Figure 4. For example, at 10 gVS / L OL, the presence of oxygen increased butyrate concentrations from a low of 5.6 g / L observed in the absence of oxygen to 5.8 g / L. This trend was even more pronounced at 15 gVS / L OL, where butyrate concentrations reached a maximum of 6.7 g / L under aerobic conditions, significantly higher than the 5.1 g / L observed in the absence of oxygen. In contrast, at the highest OL level of 25 gVS / L, peak concentrations were much lower, reaching only 1.7 g / L in the absence of oxygen and 0.7 g / L in the presence of oxygen, suggesting that very high substrate concentrations or other limiting factors may have produced an inhibitory effect at this OL level. These results may be due to anaerobic fermentation by Clostridium species, which metabolize carbohydrates into butyrate and other volatile fatty acids via the acetyl-CoA pathway. In some cases, the introduction of oxygen appears to enhance butyrate production, likely due to the activity of aerobic or facultative anaerobic bacteria that can utilize alternative metabolic pathways to produce butyrate. For example, at a low OL level (5 gVS / L), the introduction of oxygen slightly increased the maximum butyrate concentration from 3.4 g / L to 3.8 g / L, suggesting that oxygen can influence microbial kinetics and fermentation outcomes. Given the complex interplay between microbial metabolism, substrate availability, and environmental conditions, these results highlight the importance of balancing OL levels and oxygen availability for optimal butyrate production.

[0096] 2.2.4. Formation of formic acid

[0097] The generation of formic acid reveals the influence of OL concentration and oxygen supply on the degradation process of food waste. Figure 5 As shown, at all OL levels, the presence of oxygen consistently resulted in decreased formate concentrations compared to anaerobic conditions. At the lowest OL of 5 gVS / L, formate concentrations on day 8 ranged from 0.1 g / L in the absence of oxygen to 0.09 g / L in the presence of oxygen. Similarly, at the highest OL of 25 gVS / L, concentrations on day 8 ranged from 0.1 g / L in the absence of oxygen to 0.06 g / L in the presence of oxygen. However, at higher OL levels (15 gVS / L and 25 gVS / L), the effect of oxygen appeared to diminish, suggesting the potential for saturation effects or alternative degradation pathways at elevated initial formate concentrations. The interplay between initial formate concentration, oxygen availability, and degradation rate highlights the complex dynamics of formate degradation mechanisms that may exist for certain microorganisms during food waste degradation, necessitating further comprehensive investigation to elucidate the underlying chemical pathways.

[0098] 2.2.5. Propionic acid formation

[0099] Figure 6 shows that propionate levels increased in a concentration-dependent manner over 8 days under various initial concentrations and conditions (anaerobic and aerobic). At an OL of 25 gVS / L, the highest value was 1.4 g / L in the anaerobic treatment and 1.3 g / L in the aerobic treatment. Conversely, the lowest value occurred on day 1 at 5 gVS / L, with propionate concentrations of 0.10 g / L in the anaerobic treatment and 0.12 g / L in the aerobic treatment. The presence of the oxidant appeared to slightly increase propionate levels compared to non-oxidizing conditions, suggesting a potential stabilizing or enhancing effect. Overall, propionate concentrations increased over time under both conditions at all initial concentrations, indicating a time-dependent accumulation of propionate. Microbial degradation of propionate may involve multiple mechanistic pathways. In the presence of oxygen, one possible pathway involves the oxidation of propionate to form propionyl-CoA, which can undergo further oxidative decarboxylation to produce acetyl-CoA and succinate. Acetyl-CoA can then enter the TCA cycle for energy production. The presence of oxidants may promote these oxidation reactions, leading to the observed increase in propionate levels. In the absence of oxidants, alternative pathways such as fermentation or anaerobic metabolism may predominate, potentially producing different intermediates and metabolic outcomes.

[0100] 2.2.6. Total VFA production

[0101] Figure 7 shows the dynamics of total volatile fatty acid (VFA) production under various oxygen (OL) conditions, specifically focusing on the effects of the presence or absence of oxygen on five different initial OLs (5 gVS / L, 10 gVS / L, 15 gVS / L, 20 gVS / L, and 25 gVS / L). The results provide several key insights into VFA production trends and yields in these controlled environments. Starting at the lowest concentration of 5 gVS / L, total VFAs gradually increased from 3.4 g / L to 14.2 g / L under anaerobic conditions. Conversely, the presence of oxygen promoted the increase in VFA concentration, with total VFA concentrations increasing from 4.0 g / L to 16.1 g / L, suggesting that oxygen treatment may catalyze or promote more efficient VFA conversion in food waste. Similar trends were observed at an OL of 10 gVS / L, but with higher overall VFA yields. In the absence of oxygen, the final VFA concentration reached 22.2 g / L. Under aerobic treatment, VFA production peaked at 24.5 g / L on day 8. This pattern emphasizes the role of oxygen treatment in enhancing VFA production, a trend that became more pronounced at higher substrate concentrations. At an OL of 15 g VS / L, VFA concentrations under anaerobic conditions (26.4 g / L) were significantly lower than those under aerobic conditions (30.0 g / L), further confirming that oxygen treatment not only promoted VFA production but also that its effects were amplified at higher substrate concentrations. An interesting anomaly emerged at a concentration of 20 g VS / L, particularly under oxygen treatment, where VFA production initially increased from 10.2 g / L to a dramatic increase of 37.0 g / L on day 8. This suggests the existence of a threshold or critical point during anaerobic fermentation, at which oxygen and substrate concentrations act synergistically to amplify VFA production. Finally, at an OL of 25 gVS / L, the data revealed a plateau effect, with VFA production in the untreated samples decreasing slightly from a peak value, increasing from 9.6 g / L on day 1 to 24.8 g / L on day 8. However, oxygen treatment increased VFA concentrations from 10.1 g / L to 25.8 g / L. This suggests a potential saturation point or limit for oxygen catalysis at high substrate concentrations. These results demonstrate a clear dependence of VFA production on OL concentration, with the presence of oxygen having a significant effect. Oxygen treatment consistently increased VFA production at all OL concentrations, with the effect becoming more pronounced at intermediate concentrations, but potentially reaching a saturation point at an OL of 25 gVS / L. This intricate interplay between substrate concentration, the presence of low levels of oxygen, and VFA production highlights a complex biochemical mechanism, potentially involving enhanced oxidative pathways or increased substrate utilization in the presence of oxygen.

[0102] 2.3 Ammonia nitrogen and COD concentrations at different organic loadings during anaerobic digestion of food waste under aerobic and anaerobic conditions

[0103] Figure 8 shows data from monitoring ammonium nitrogen concentrations under aerobic and anaerobic conditions at various OL concentrations (5 gVS / L, 10 gVS / L, 15 gVS / L, 20 gVS / L, and 25 gVS / L), revealing the complex interplay of biological and chemical processes influencing nitrogen dynamics in these systems. At a concentration of 5 gVS / L, the presence of oxygen appeared to slightly reduce ammonium nitrogen concentrations compared to anaerobic conditions, with the lowest concentration observed being 0.07 g / L under aerobic conditions on day 8. This suggests that aerobic conditions may enhance nitrification—the process by which nitrifying bacteria convert ammonia to nitrate—thus reducing ammonia levels. As substrate concentrations increased to 10 gVS / L and 15 gVS / L, an overall increasing trend in ammonium nitrogen concentrations was observed, particularly under anaerobic conditions. This may be due to the increased availability of organic nitrogen compounds at higher substrate concentrations, which undergo ammonification, converting organic nitrogen to ammonia. The observed fluctuations in ammonium nitrogen concentrations, particularly under aerobic conditions, may reflect a dynamic balance between ammonification and nitrification. Ammonium nitrogen concentrations peaked at OL of 20 gVS / L and 25 gVS / L, with maximum concentrations of 0.45 g / L and 0.50 g / L, respectively, observed in the absence of oxygen on day 8. These elevated levels may indicate saturation or inhibition of the nitrification process, either due to higher organic loadings that inhibit the ability of nitrifying bacteria or due to the accumulation of inhibitory substances that hinder bacterial activity.

[0104] As shown in Figure 9, the COD values ​​monitored at different substrate concentrations provide insight into the oxidation behavior of organic matter in the samples. Under 5 g VS / L, COD values ​​increased gradually from 18.6 g / L to 28.2 g / L in the absence of oxygen. Under aerobic conditions, COD concentrations increased from 18.3 g / L to 26.7 g / L, a decrease compared to the absence of oxygen. This indicates that oxygen has little effect on the oxidation process at lower OL concentrations. At 10 g VS / L, the increase in COD values ​​in the absence of oxygen was more pronounced, increasing from 33.0 g / L to 47.3 g / L on day 1. In contrast, under aerobic conditions, COD values ​​increased from 18.4 g / L to 47.1 g / L, nearly identical to the COD concentration in the absence of oxygen. Oxygen significantly accelerates the oxidation process, as confirmed by the results at an OL concentration of 15 g VS / L. In the absence of oxygen, the COD value increased to 33.5 g / L and then increased to 71.28 g / L on the 8th day. In the presence of oxygen, it increased significantly from 29.5 g / L to 71.9 g / L. At a concentration of 20 gVS / L, the COD concentrations in the absence and presence of oxygen were almost the same, starting from 36.6 g / L in the absence of oxygen and reaching a maximum value of 104.8 g / L, indicating a large organic load. In the presence of oxygen, the initial value was also 36.5 g / L, but the final value was slightly lower at 104.4 g / L. The most significant increase in COD was observed at the highest OL concentration of 25 gVS / L. In the absence of oxygen, the COD value started from 43.0 g / L and increased to 109.2 g / L on the 8th day. In the presence of oxygen, the initial value was 40.8 g / L and peaked at 111.8 g / L, indicating that oxygen's effect on the oxidation of organic compounds is more pronounced at higher concentrations. Previous studies have shown that these observations highlight a clear trend: COD values ​​increase with increasing substrate concentration, indicating higher organic loads and a greater demand for oxygen for effective oxidation. The presence of oxygen generally increases COD, suggesting that oxygen facilitates more complete oxidation of organic matter, highlighting its importance in environmental and wastewater management strategies aimed at mitigating the impacts of organic pollutants.

[0105] 2.4 Schizochytriumlimacinum SR21 uses VFA to produce lipid-rich single-cell protein

[0106] 2.4.1 Effects of different initial pH conditions on VFA utilization by Schizochytrium

[0107] pH is one of the important factors affecting microbial growth and metabolite synthesis. On the one hand, pH changes the charge of the plasma membrane in the cell, thereby affecting the microbial absorption of nutrients and, in turn, the growth of the bacteria. On the other hand, the activity of enzymes in microorganisms is affected by pH, thereby affecting the metabolic regulation in the organism and, in turn, the synthesis of products. In order to find the pH value suitable for bacterial growth and product synthesis, Schizochytriumlimacinum The results of the initial pH optimization of SR21 in VFA fermentation culture provided valuable insights into the interactions between changes in pH levels, VFA consumption, and microbial growth.

[0108] The data present a systematic evaluation of lactate and VFA concentrations (including formate, acetate, propionate, and butyrate) under conditions varying in initial pH from 6 to 9. A detailed study of the utilization of lactate and volatile fatty acids by Schizochytrium sp. SR21 at various initial pH values ​​provided insight into the metabolic preferences and efficiency of the fungus. As shown in Figure 10, this study demonstrates that an initial pH of 6-8 favors the consumption of organic acids, highlighting the optimal environment for acid metabolism. From 0 to 48 hours, the levels of formate, acetate, propionate, and butyrate showed a continuous downward trend, and within 72 hours, all of these acids were completely consumed. Lactic acid consumption, however, exhibited a reverse trend. During the initial 0-48 hours, the consumption of other acids inhibited lactate uptake, resulting in lower lactate consumption. After 72 hours, lactate content continued to decline, but some lactate remained in the fermentation broth after 144 hours of fermentation. The consumption of organic acids at an initial pH of 7 suggests that neutral or slightly acidic conditions may enhance the activity of enzymes involved in breaking down these acids. Interestingly, at an initial pH of 5, there was almost no change in the pH of the culture broth throughout the fermentation ( Figure 11 This is likely due to acid stress on the cells, which completely inhibits their growth or even causes them to die, resulting in the VFA in the fermentation broth not being utilized, and thus the pH of the fermentation broth remained unchanged. When the initial pH was 9, a decrease in the pH of the fermentation broth was detected within the first 24 hours. This is likely due to the cells eliminating acidic small molecules and carbon dioxide in the cell body in order to adapt to the environment, resulting in a decrease in the pH of the fermentation broth ( Figure 11 In addition, within 72 h, acetic acid and butyric acid were basically not consumed, and only a small amount of formic acid, propionic acid and lactic acid were consumed ( Figure 10d), which is exactly the same as the pH change in the fermentation broth. This indicates that in the early stages of organic acid utilization, cell growth is inhibited to withstand the harsh external environment, resulting in a growth stagnation period. In addition, the highest biomass and protein yields detected at initial pH 6 and 7 reached 16.42 g / L and 6.13 g / L, respectively, indicating that pH 6 and 7 are the optimal conditions for acid utilization ( Figure 11 Similarly, previous studies have investigated the effects of culture conditions, pH, carbon source, and nitrogen source on Schizochytrium S31 and found that at an initial pH of 7, Schizochytrium S31 can accumulate up to 40% biomass and 13% (w / w) DHA lipids. However, when examining the lipid content and fatty acid composition within the cells, it was found that the lipid content significantly increased at an initial pH of 9, reaching a maximum of 21.58%. However, the content of unsaturated fatty acids, primarily DHA, decreased, while the content of saturated fatty acids, primarily C16:0, increased ( Figure 11 This suggests that there may be a trade-off or optimization requirement between achieving metabolic efficiency and supporting cell growth, which may have an impact on the high-density growth of microbial cells and efficient substrate utilization.

[0109] Trends in VFA consumption throughout the study suggest that pH optimization strategies can enhance metabolic activity and biomass production in culture. This is particularly evident in the poor performance of the initial pH of 9, both in terms of acid utilization and cell growth. This pH level has a significant inhibitory effect on cell growth, highlighting the importance of precise pH control in microbial culture environments. While an initial pH of 7 is believed to be beneficial for the metabolic processes of Schizochytrium, its preference for slightly acidic conditions allows for maximum growth, which provides valuable insights for biotechnological applications. Overall, this study highlights the critical role of pH in influencing microbial metabolism and growth dynamics, with this pH balance helping to effectively decompose acids and support cell growth, highlighting the importance of controlled environmental conditions in industrial bioapplications.

[0110] 2.4.2 Effects of different VFA concentrations on the growth of Schizochytrium SR21

[0111] To further investigate VFA utilization by Schizochytrium, the utilization of lactate, formate, acetate, propionate, and butyrate by Schizochytrium was monitored at different initial concentrations (25%, 50%, 75%, and 100% VFA), as shown in Figure 12. VFA utilization by Schizochytrium SR21 provides valuable insights into the metabolic capabilities and preferences of this microorganism and reveals how microbial strains adapt to and metabolize different VFAs.

[0112] Schizochytrium utilizes lactate, formate, acetate, propionate, and butyrate differently under different VFA concentrations. At 25% and 50% VFA, all acids except lactate were completely consumed within 48 hours of fermentation. This indicates that lower substrate concentrations allow for better cellular catabolism and utilization, supporting rapid microbial growth. At 75% and 100% VFA, formate, propionate, and butyrate were all consumed within 48 hours, while acetate was consumed within 144 hours of fermentation. In contrast, lactate degradation was slower, with some acid remaining unconsumed even after fermentation. This suggests that enzymes are stressed by high lactate concentrations, exhibiting lower substrate affinity or requiring more complex and energy-intensive pathways to degrade them. Previous studies have shown that high substrate concentrations can inhibit the activity of key enzymes such as glycerol kinase and glycerol-3-phosphate dehydrogenase. Therefore, further optimization and enhancement of microbial lactate degradation are needed in future experiments. In addition, during the fermentation process, the pH value in the fermentation liquid rises rapidly in the early stage, but rises slowly in the late stage of fermentation, and the final pH value reaches about 9.2 ( Figure 13 (a) This change is associated with the degradation of VFAs, suggesting that the strain was rapidly utilizing these acids as a carbon flux and energy source. The increase in pH over time further supports the hypothesis that acid degradation occurred, leading to a decrease in environmental acidity. Figure 13 Figure b shows the biomass and protein content under different VFA concentrations. Interestingly, as the VFA concentration increases, the biomass of Schizochytrium gradually increases, but the protein content gradually decreases. Under 25% VFA conditions, the protein content of Schizochytrium can reach 49.35%, which is 11.22% higher than that under 100% VFA conditions; while under 100% VFA conditions, the biomass and protein yield of Schizochytrium are significantly increased, reaching a maximum of 15.29 g / L and 5.83 g / L, respectively. Compared with 25% VFA conditions, the biomass and protein yield increased by 121.59% and 71.47%, respectively. It is speculated that the total protein content may be reduced due to the increase in the carbon-nitrogen ratio. Similarly, previous studies have shown that when studying the use of microalgae to convert rice hydrolysate (BRH) into protein, it was found that a low C / N ratio is beneficial to protein synthesis in microalgae cells. At the same time, the oil content and fatty acid composition and content at different concentrations were also detected, but there was no significant change ( Figure 13 (c)

[0113] Therefore, in this study, while higher VFA concentrations favored biomass production, an increase in the carbon-to-nitrogen ratio compromised microbial single-cell accumulation. This insight is crucial for scaling up this process, as it demonstrates the need for precise control of VFA concentrations to avoid inhibitory effects on microbial metabolism. In summary, in subsequent experiments, we will select a fermentation medium containing 100% VFA at pH 7 for Schizochytrium cultivation.

[0114] 2.4.3 Expression of Lactate Dehydrogenase / Lactate Oxidase Genes in Schizochytrium Improves Lactic Acid Utilization

[0115] Previous studies have shown that when Schizochytrium utilizes VFAs, lactate is not fully utilized. This suggests that low lactate dehydrogenase activity in Schizochytrium, or that high lactate concentrations are causing stress in the organism, leads to low lactate utilization. Genes related to lactate utilization have been extensively studied in various microorganisms. These genes encode enzymes that are primarily involved in the lactate metabolism pathway, including the oxidation and dehydrogenation of lactate, and its subsequent conversion to other metabolites. Among them, lactate dehydrogenase (LDH) genes are the most common core genes involved in lactate metabolism, catalyzing the reversible conversion of lactate to pyruvate. LDHs can be divided into two subfamilies (d-LDH and l-LDH) depending on the substrate. Furthermore, the enzyme encoded by the lactate oxidase (LOX) gene is a flavin mononucleotide (FMN)-dependent flavoenzyme that can directly oxidize lactate to pyruvate in the presence of oxygen (O2) without the cofactor NAD. + The existence of Figure 14 Since LOX is tightly bound to its cofactor FMN, it does not require additional FMN during biotransformation, thus having great potential and application value in industrial applications. LOX has been found in many microorganisms, including Pediococcus sp., Aerococcus viridans , Streptococcus iniae , Geotrichumcandidum and Lactococcus lactis Among the various LOX, A. viridans AvLOX (AvLOX) has the highest activity toward lactate and has been intensively studied for use in biosensors to measure lactate concentrations in blood or other body fluids.

[0116] In order to enable Schizochytrium to better utilize the byproduct lactic acid produced by food waste, the present invention expressed endogenous LDH and the lactic acid from Aerococcus viridans ( A. viridans) to enhance the utilization of lactic acid by Schizochytrium. The constitutive promoter PA was used to express d-LDH, l-LDH and SpLOX genes in cells. The pK2-PE-NPTII vector only contains resistance genes and GUS genes, which are expressed in Schizochytrium to exclude the influence of the insertion site on gene expression. Verification by PCR and GUS staining showed that the genes were successfully inserted into the Schizochytrium genome. Among them, the recombinant strains d-LDH, l-LDH and PE amplified the GUS-NPTII gene fragment (2661bp) by PCR, while the recombinant strain spLOX-2 successfully amplified the SpLOX gene fragment (1122bp) by PCR ( Figure 15 To evaluate the lactate utilization capacity of the overexpressing strains, the recombinant strains (d-LDH, l-LDH, SpLOX, PE) and wild-type SR21 were fermented in VFAs fermentation medium. Figure 16 The results show the VFA consumption of the recombinant strains and the wild-type SR21. Except for the wild-type strain, all recombinant strains consumed formic acid, acetic acid, propionic acid, and butyric acid within 72 hours. Surprisingly, after the fermentation, the recombinant strain spLOX-2 had the least residual lactic acid, and its lactic acid consumption increased by 8.31% compared to the wild-type. Figure 17 b), which may be due to the higher activity of lactate oxidase than lactate dehydrogenase, which improves the utilization of lactate. At the same time, due to the increased utilization of lactate by the recombinant strain spLOX-2, its biomass was significantly higher than that of the wild-type strain, reaching a maximum of 17.46 g / L ( Figure 17 c), which was significantly increased by 14.16% compared to the wild type. In addition, the conversion rate of the recombinant strain was increased by 3.08% compared to the wild type. The protein yield of the recombinant strain was 6.53 g / L, which was increased by 12% compared to the wild type. Surprisingly, the oil content of the recombinant strain reached 14.87%, which was significantly increased by 107.1% compared to the wild type ( Figure 17 (d) It is speculated that lactate is decomposed into pyruvate by lactate oxidase, and the increased utilization of lactate leads to an increase in pyruvate content. The fate of pyruvate in microorganisms is mainly nitrogen metabolism, amino acid metabolism, and conversion to acetyl-CoA by propionate dehydrogenase to enter the fatty acid synthesis pathway, or decomposition into oxaloacetate by pyruvate carboxylase to enter the TCA cycle ( Figure 14Schizochytrium is an oil-producing microorganism with strong fatty acid metabolism. Increased pyruvate levels are likely to increase acetyl-CoA levels. Acetyl-CoA is a key precursor in the lipid biosynthesis pathway. Increased acetyl-CoA upregulates this pathway, leading to increased lipid content in the recombinant spLOX-2 strain. However, the specific mechanism requires further investigation.

[0117] At the same time, the Schizochytrium spLOX-2 strain was deposited, and the classification of the Schizochytrium spLOX-2 strain was named: Schizochytrium Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. spLOX-2 is deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) with the deposit number of CGMCC No. 41623 and the deposit date of November 11, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0118] Therefore, the expression of lactate oxidase in Schizochytrium improves the utilization of lactic acid by Schizochytrium, enabling Schizochytrium to efficiently convert VFA from food waste into oil-rich single-cell protein, providing a new path for a sustainable bio-circular economy model. However, due to the complexity of the process for treating food waste, further research will focus on expanding the scale of the process and improving the economic feasibility of the technology in industrial applications. In short, these research results provide a new way for further exploration of Schizochytriumlimacinum SR21 lays the foundation for the mechanistic pathway of VFA utilization, which has important implications for biotechnological applications such as value-added chemical production and microbial fermentation processes.

[0119] 3 Conclusion

[0120] In this study, the innovative combination of micro-aeration and thermal pretreatment significantly enhanced enzymatic hydrolysis of food waste, thereby increasing VFA production. Furthermore, fermentation optimization revealed the critical role of pH in maximizing VFA utilization and microbial growth in Schizochytrium, with an initial pH of 7 being found to favor metabolic processes. This pH balance facilitates efficient acid decomposition and supports cell growth, highlighting the importance of controlled environmental conditions in industrial bioapplications. Furthermore, the study demonstrated that while higher VFA concentrations favor biomass production, an increase in the carbon-to-nitrogen ratio compromises the accumulation of microbial single-cell content. This insight is crucial for scaling up the process, as it demonstrates the need for precise control of VFA concentration to avoid inhibitory effects on microbial metabolism. Furthermore, expression of lactate oxidase in Schizochytrium significantly enhanced lactate utilization, resulting in a significant increase in biomass and the production of single-cell proteins rich in high-value lipids. This study demonstrates the significant potential of Schizochytrium for VFA utilization from food waste. Therefore, this research provides an excellent foundation for developing a sustainable bioeconomy model in which food waste is not only disposed of but also converted into economically valuable products. This not only helps reduce environmental pollution but also contributes to resource reuse, in line with global sustainable development goals. Future research will aim to improve and scale up the process and explore its commercial viability on a larger scale.

[0121] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and exemplary embodiments. They can be applied to a variety of fields suitable for the embodiments of the present invention. Those skilled in the art will readily realize further modifications. Therefore, without departing from the general concept defined by the claims and their equivalents, the embodiments of the present invention are not limited to the specific details and embodiments shown and described herein.

Claims

1. A Schizochytrium strain that efficiently converts food waste into single-cell protein, characterized in that: The Schizochytrium strain is Schizochytrium ( Schizochytrium sp. ) spLOX-2, the classification name of the Schizochytrium spLOX-2 strain is: Schizochytrium Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. spLOX-2 is deposited in the General Microbiology Center of China Culture Collection of Microorganisms, with the deposit number: CGMCC No.41623, the deposit date: November 11, 2024, and the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Schizochytrium strain according to claim 1 in food waste treatment and single-cell protein production.

3. A method for efficiently converting food waste into single-cell protein, characterized in that: The steps include: 1) Fermenting food waste to produce volatile fatty acids through micro-aeration and thermal pretreatment; 2) 100% volatile fatty acids produced in step 1) are used as a carbon source to culture Schizochytrium, and the initial pH is adjusted to 6-7. After several days of culture, single-cell protein is obtained from the cultured bacteria. The Schizochytrium is Schizochytrium Schizochytrium limacinum SR21 or Schizochytrium Schizochytrium sp. spLOX-2.

4. The method for efficiently converting food waste into single-cell protein according to claim 3, wherein: In step 1), fermenting food waste to produce volatile fatty acids includes the following steps: The food waste is dried and made into food waste powder. Then, the food waste powder, the original inoculum seed liquid and water are placed in a sealed container, and the air is removed to form an anaerobic environment. Then, oxygen is injected into the sealed container and anaerobic digestion is carried out under micro-aeration conditions for 1 to 8 days to obtain a fermented product of the food waste and harvest the fermentation liquid.

5. The method for efficiently converting food waste into single-cell protein according to claim 4, wherein: In step 1), in the anaerobic digestion, the initial organic load (OL) of the fermentation product is 5-25 gVS / L, and 6 mL / g VS of oxygen is injected.

6. The method for efficiently converting food waste into single-cell protein according to claim 3, wherein: In step 2), the Schizochytrium spLOX-2 is Schizochytrium spLOX-2, and the classification name of the Schizochytrium spLOX-2 strain is: Schizochytrium spLOX-2 Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. spLOX-2 is deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) with the deposit number of CGMCC No. 41623 and the deposit date of November 11, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

7. The method for efficiently converting food waste into single-cell protein according to claim 3, wherein: In step 2), the volatile fatty acids produced in step 1) are used as a carbon source by adding a fermentation medium for Schizochytrium to the fermentation liquid of the food waste fermentation in step 1) to form a final fermentation medium, and culturing Schizochytrium in the final fermentation medium.

8. The method for efficiently converting food waste into single-cell protein according to claim 7, wherein: The fermentation medium for Schizochytrium contains the following components at the following concentrations: 5 g / L yeast extract, 0.3 g / L NaCl, 1 g / L K2SO4, 0.1 g / L KH2PO4, 4 g / L MgSO4·7H2O, and 0.05 g / L CaCl2.

9. The method for efficiently converting food waste into single-cell protein according to claim 4, wherein: In step 1), the ratio of food waste powder to original inoculum seed solution was 1:10 (v / v).

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