Schizochytrium limacinum strain for efficiently converting kitchen waste to produce single-cell protein and application of schizochytrium limacinum strain

Abstract

The invention discloses a schizochytrium limacinum strain for efficiently converting kitchen waste to produce single-cell protein and application of the schizochytrium limacinum strain, and belongs to the technical field of biological engineering. The preservation number of the schizochytrium sp LOX-2 is CGMCC (China General Microbiological Culture Collection Center) No. 41623. The invention also discloses a method for producing single-cell protein by efficiently converting kitchen waste. The method comprises the following steps: fermenting food waste to produce volatile fatty acid; and culturing schizochytrium limacinum by using the strain as a carbon source to obtain single-cell protein. According to the recombinant strain spLOX-2, the utilization of lactic acid is improved, the biomass and the protein yield of the recombinant strain spLOX-2 are respectively improved by 14.19% and 12% compared with those of a wild strain, and the grease content is obviously improved by 107.1%. According to the invention, a new way is provided for a sustainable biological economic mode, wastes are converted into valuable biological chemicals, and a solid foundation is laid for expanding the scale of the process and the economic feasibility of the process in industrial application.

Description

Technical Field

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

[0002] Facing the global protein shortage problem, biomanufacturing technology is considered one of the important solutions. Biomanufacturing technology uses microorganisms, enzymes, or cell factories to synthesize target products through biotransformation means, showing great potential in fields such as food, medicine, and chemical engineering. In recent years, microbial-based protein production, such as single-cell protein (SCP), is considered an effective way to replace traditional protein sources (such as animal and plant proteins). However, traditional biomanufacturing still faces a key bottleneck - the acquisition of low-cost raw materials. Currently, most biomanufacturing processes rely on expensive sugars, starches, or other fermentable organic substances as raw materials, which not only increases production costs but also intensifies the competition for food resources. In addition, traditional microbial cultivation usually requires a complex pretreatment process with high process costs, making it difficult to maintain competitiveness in industrial applications. Therefore, finding cheap, efficient, and sustainable raw material sources is an important breakthrough point in the field of biomanufacturing.

[0003] Food waste, as a potential raw material source for biomanufacturing, has the advantages of rich resources and low costs. Its main components include carbohydrates, proteins, fats, and other organic substances, with high biodegradation potential. However, due to the complex composition of food waste, there are many technical difficulties in direct utilization, such as contamination by miscellaneous bacteria and low conversion efficiency. Therefore, how to efficiently convert food waste into raw materials available for biomanufacturing is the key issue in current research.

[0004] In recent years, the rapid development of synthetic biology has provided a new technical path for the high-value utilization of food waste. By using synthetic biology means to transform microorganisms, microorganisms can efficiently convert specific substrates to produce target biological products. These products are widely used in various industries such as food, cosmetics, chemicals, and textiles, and can realize the reuse and value addition of waste. Based on the "circular bioeconomy model" of microbial-based waste biorefineries, by combining the biotransformation stage with microbial growth to produce high-value products and renewable biofuels, it aims to recycle and reuse waste streams. However, due to low biomass production, high downstream processing costs, and other capital requirements, microbial cell-based factories cannot produce economically and sustainably viable biochemicals. Therefore, in the microbial fermentation process, only the co-production of high biomass and various value-added chemicals can achieve the maximum value-added benefit of waste streams. Summary of the Invention

[0005] One object of the present invention is to solve at least the above problems and / or deficiencies and provide at least the advantages 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 as follows: A Schizochytrium strain that can efficiently convert food waste into single-cell protein, wherein the Schizochytrium strain is Schizochytrium spLOX-2, and the taxonomic name of the Schizochytrium spLOX-2 strain is: Schizochytrium Schizochytrium sp. , the strain Schizochytrium Schizochytrium sp. spLOX-2 is deposited in the China General Microbiological Culture Collection Center (abbreviated as CGMCC), the deposit number is: CGMCC No. 41623, the deposit time is: November 11, 2024, and the address of the deposit unit is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Application of the described Schizochytrium strain in food waste treatment and single-cell protein production.

[0009] A method for efficiently converting food waste into single-cell protein, comprising the following steps: 1) Ferment food waste to produce volatile fatty acids VFA; 2) Use the volatile fatty acids produced in step 1) as a carbon source to culture Schizochytrium. After culturing for several days, obtain single-cell protein from the cultured bacteria. More preferably, in step 2), culture for 6 days.

[0010] Preferably, in the method for efficiently converting food waste into single-cell protein, in step 1), fermenting food waste to produce volatile fatty acids includes the following steps: Take food waste, dry it to make food waste powder, then take food waste powder, original inoculum seed liquid and water and place them in a sealed container, exclude air to form an anaerobic environment, and then inject oxygen into the sealed container, and carry out anaerobic digestion for 1-8 days under micro-aeration conditions to obtain the fermented product of food waste, and harvest the fermentation broth.

[0011] Preferably, in the method for efficiently converting food waste into single-cell protein, in step 1), in the anaerobic digestion, the initial OL of the fermented product is 5-25 gVS / L, that is, each liter of the fermented product contains 5 to 25 grams of food waste volatile solids VS, and 6 mL / g VS of oxygen is injected, that is, 6 milliliters of oxygen need to be injected per gram of VS (usually measured under standard temperature and pressure). More preferably, the fermentation temperature is 37°C.

[0012] Preferably, in the method for efficiently converting food waste into single-cell protein, the following steps are further included: The lactic acid oxidase LOX gene from Aerococcus viridans or the d-LDH gene and l-LDH gene of Schizochytrium limacinum are respectively transformed into Schizochytrium limacinum after codon optimization to obtain three recombinant Schizochytrium limacinum strains. In step 2), any one or several of the three recombinant Schizochytrium limacinum strains are used as the Schizochytrium limacinum.

[0013] Preferably, in the method for efficiently converting food waste into single-cell protein, in step 2), the Schizochytrium limacinum is Schizochytrium spLOX-2, and the taxonomic name of the Schizochytrium spLOX-2 strain is: Schizochytrium Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. spLOX-2 is deposited in the China General Microbiological Culture Collection Center (abbreviation: CGMCC), the deposit number is: CGMCC No. 41623, the deposit time is: November 11, 2024, and the address of the deposit unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0014] Preferably, in the method for efficiently converting food waste into single-cell protein, in step 2), the way of using the volatile fatty acids produced in step 1) as a carbon source is to add a fermentation medium for Schizochytrium limacinum to the fermentation broth of food waste fermentation in step 1), adjust the initial pH to 6-9 to form a final fermentation medium, and culture Schizochytrium limacinum with the final fermentation medium.

[0015] Preferably, in the method for efficiently converting food waste into single-cell protein, the fermentation medium for Schizochytrium limacinum 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, 0.05 g / L CaCl2.

[0016] Preferably, in the method for efficiently converting food waste into single-cell protein, in step 1), the ratio of food waste powder to the original inoculum seed liquid is 1:10 (v / v).

[0017] Preferably, in the method for efficiently converting food waste into single-cell protein, the Schizochytrium limacinum used is Schizochytrium SR21. More preferably, the culture temperature is 28°C. Adjust the initial pH to 6-7.

[0018] The present invention has at least the following beneficial effects: With the continuous increase in the generation of municipal solid waste (MSW), especially food waste (FW), the world is facing severe environmental challenges and resource management problems. The present invention utilizes micro-aeration / thermal pretreatment to improve the biodegradability of FW, converts FW into valuable volatile fatty acids (VFA), and then Schizochytrium uses VFA to produce oil-rich single-cell protein, thereby realizing the value-added utilization of FW. The results show that under oxygen-containing conditions with an organic loading rate of 20 gVS / L, the VFA yield increases significantly, reaching 37.0 g / L, demonstrating that oxygen treatment can effectively improve the VFA yield. In addition, the present invention also finds that Schizochytrium SR21 has good utilization ability for VFA. This discovery opens up a new idea for the microbial utilization of FW. Experiments show that under the conditions of pH 7 and 100% VFA, this strain shows strong VFA consumption ability, and the concentration of short-chain VFA decreases significantly over time, indicating its efficient metabolic process. Further, by expressing exogenous lactate oxidase, the lactate consumption ability is significantly improved, and the biomass, oil content, and protein yield of the recombinant strain increase significantly, reaching up to 17.46 g / L, 14.87%, and 6.53 g / L respectively. Among them, the biomass and protein yield are increased by 14.19% and 12% respectively compared with the wild type, while the oil content is significantly increased by 107.1%. The present invention provides a new path for the sustainable bio-economic model, proposes a sustainable and efficient protein production path, aims to solve the global protein shortage problem, and promotes the resource utilization of food waste. In this model, waste is converted into valuable biochemicals, which not only conforms to the concepts 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.

[0019] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a design schematic diagram of the experimental device in the present invention.

[0021] Figure 2 It is a graph showing the change of lactate concentration under different organic loads under anaerobic pretreatment conditions in the present invention.

[0022] Figure 3 It is a graph showing the change of acetic acid concentration under different organic loads under anaerobic pretreatment conditions in the invention.

[0023] Figure 4 It is a graph showing the change of butyric acid concentration under different organic loads under anaerobic pretreatment conditions in the present invention.

[0024] Figure 5 It is a graph showing the change of formic acid concentration under different organic load conditions with or without anaerobic pretreatment in the present invention.

[0025] Figure 6 It is a graph showing the change of propionic acid concentration under different organic load conditions with or without anaerobic pretreatment in the present invention.

[0026] Figure 7 It is a graph showing the change of total VFA concentration under different organic load conditions with or without oxygen pretreatment in the present invention.

[0027] Figure 8 It is a graph showing the change of ammonium nitrogen concentration under different organic load conditions with or without anaerobic pretreatment in the present invention.

[0028] Figure 9 It is a graph showing the change of chemical oxygen demand (COD) concentration under different organic load conditions with or without anaerobic pretreatment in the present invention.

[0029] Figure 10 Under different initial pH conditions in the present invention Schizochytrium limacinum Graph of the consumption of VFA during the fermentation culture of SR21 using VFA. a is the consumption of VFA by Schizochytrium under the initial pH of 6; b is the consumption of VFA by Schizochytrium under the initial pH of 7; c is the consumption of VFA by Schizochytrium under the initial pH of 8; d is the consumption of VFA by Schizochytrium under the initial pH of 9.

[0030] Figure 11 Under different initial pH conditions in the present invention Schizochytrium limacinum Graph of fermentation parameters for the production of single cell protein by SR21 using VFA fermentation culture. a is the change of pH in the fermentation broth during the cultivation of Schizochytrium using VFA under different initial pH conditions; b is the biomass, protein content and protein yield detected after the fermentation of Schizochytrium using VFA under different initial pH conditions; c is the oil content, fatty acid composition and content detected after the fermentation of Schizochytrium using VFA under the initial pH condition.

[0031] Figure 12 Under different concentrations of VFA conditions in the present invention Schizochytrium limacinumFigure showing the consumption of VFA during the fermentation culture of SR21 using VFA. a shows the consumption of VFA by Schizochytrium sp. under 25% VFA condition; b shows the consumption of VFA by Schizochytrium sp. under 50% VFA condition; c shows the consumption of VFA by Schizochytrium sp. under 75% VFA condition; d shows the consumption of VFA by Schizochytrium sp. under 100% VFA condition.

[0032] Figure 13 Under different concentrations of VFA conditions in the present invention Schizochytrium limacinum Figure of fermentation parameters for the production of single cell protein by SR21 using VFA fermentation culture. a shows the pH change of the fermentation broth during the cultivation of Schizochytrium sp. using VFA under different concentrations of VFA; b shows the biomass, protein content and protein yield detected after the fermentation of Schizochytrium sp. using VFA under different concentrations of VFA; c shows the oil content, fatty acid composition and content detected after the fermentation of Schizochytrium sp. using VFA under different concentrations of VFA.

[0033] Figure 14 Partial metabolic pathway diagram in Schizochytrium sp. of the present invention.

[0034] Figure 15 Diagram of plasmid construction and recombinant strain verification used in Schizochytrium sp. of the present invention. a shows the plasmid construction of overexpressed genes in Schizochytrium sp.; b shows the PCR verification of recombinant strains; c shows the qualitative detection of the expression of the reporter gene β-glucosidase (GUS) in recombinant strains.

[0035] Figure 16 Figure showing the consumption of VFA during the fermentation culture of Schizochytrium sp. using VFA in the present invention. a shows the consumption of VFA by the recombinant strain d-LDH; b shows the consumption of VFA by the recombinant strain l-LDH; c shows the consumption of VFA by the recombinant strain spLOX; d shows the consumption of VFA by the recombinant strain PE; e shows the consumption of VFA by the wild-type strain SR21.

[0036] Figure 17This is a fermentation parameter diagram for the production of single-cell protein by Schizochytrium using VFA fermentation. a shows the pH change of the fermentation broth during the cultivation of recombinant and wild-type Schizochytrium strains using VFA; b shows the total VFA consumption and conversion rate detected after the fermentation of recombinant and wild-type strains using VFA; c shows the biomass, protein content, and protein yield detected after the fermentation of recombinant and wild-type strains using VFA; d shows the oil content, fatty acid composition, and content detected after the fermentation of recombinant and wild-type strains using VFA.

[0037] The taxonomic naming of the Schizochytrium spLOX-2 strain is: Schizochytrium Schizochytrium sp. , the strain Schizochytrium Schizochytrium sp. spLOX-2 was deposited in the China General Microbiological Culture Collection Center (abbreviated as CGMCC), with the deposit number: CGMCC No. 41623, the deposit date: November 11, 2024, and the address of the deposit unit: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed implementation methods

[0038] The following further elaborates on the present invention in conjunction with the attached drawings, enabling those skilled in the art to implement it with reference to the written description.

[0039] It should be understood that terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0040] It should be noted that the experimental methods described in the following implementation examples are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0041] Due to reasons such as fast growth rate, high cell density, and shear force tolerance, Schizochytrium has become a promising industrial microorganism. It can utilize various organic carbon sources (such as glucose, glycerol, volatile fatty acid VFA, and waste fermentation broth) for metabolism, synthesizing abundant polyunsaturated fatty acids (such as DHA) and a small amount of high-value products such as squalene and astaxanthin, providing additional value for its application in functional foods and feeds. However, the production cost of glucose is relatively high, and it can account for up to 80% of the production cost when using glucose as a carbon source. Compared with glucose or other carbon sources, using volatile fatty acids can significantly reduce the cost of microbial fermentation. In addition, since the metabolic pathway of VFA is shorter than that of sugar substrates, it improves the conversion of VFA and the synthesis of secondary metabolites. Therefore, using Schizochytrium to treat VFA in food waste fermentation broth can not only reduce production costs but also achieve resource recycling.

[0042] In the present invention, micro-aeration and thermal pretreatment are combined to enhance enzymatic hydrolysis to improve VFA production efficiency and accelerate the AD (anaerobic digestion) rate. This combined method makes full use of the advantages of each pretreatment method to maximize the effectiveness of the whole process. The micro-aeration pretreatment process involves exposing the microbial part to oxygen under moderate operating conditions to accelerate the hydrolysis stage by promoting microbial activity and cell growth. Thermal pretreatment is commonly used because it is cost-effective and requires fewer chemicals than other methods. Studies have shown that thermal pretreatment can increase the yield of VFA and the AD rate.

[0043] 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 the AD rate; (2) to study the effect of organic load changes on VFA production during the AD batch process; (3) to explore the feasibility of Schizochytrium utilizing VFA from food waste and enable Schizochytrium to effectively utilize VFA as a substrate to produce single-cell protein rich in high-value oil by expressing an exogenous lactate oxidase gene. By combining AD with microbial fermentation, this study on FW treatment provides a new strategy for the successful development of various value-added chemicals.

[0044] 1 Materials and methods 1.1 Feedstock and inoculum FW was collected from the cafeteria of the Tianjin Institute of Industrial Biotechnology, China, dried in an oven, ground with a high-speed grinder (Beijing Starstone Lihe), and then added to the fermentation system. Biogas slurry (BS) and 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 and maintained for 25 minutes, and then inoculated at a ratio of 1:10 (v / v).

[0045] 1.2 Micro-aeration / thermal pretreatment and organic load (OL) Before adding to the reactor, food waste (FW) was dried in an oven at 100 °C for 72 h and ground into powder using a high-speed grinder. Its composition is shown in Table 2. After thermal pretreatment, FW was placed in a 1 L bottle for oxygen pretreatment. During the pretreatment, 10 g of FW, 20 mL of the original inoculum seed solution, and deionized water were mixed to form a solution with a total volume of 100 mL. Then the bottle was sealed with a rubber stopper and flushed with N2 for 3 min to expel all the air in the bottle. After all the air in the bottle was expelled, 6 mL / g VS of 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 without oxygen pretreatment as controls (labeled as no oxygen pretreatment). During oxygen pretreatment, all bottles were placed in a shaker at 37 °C and 100 rpm for 24 h.

[0046] In this experiment, five different organic loads (OLs) of 5, 10, 15, 20, and 25 VSg / L were used for optimization. Each OL experiment had three replicate treatments with aerobic and anaerobic pretreatments to evaluate the effect of oxygen on various OLs and VFA production. At the beginning of the experiment, the pH was set at 7.0 and the temperature was kept constant at 37 °C during the experiment. The experiment lasted for 8 days to finally determine the optimal organic loading rate (OLR) to maximize VFA production.

[0047] 1.3 Analytical methods The total solids (TS) and volatile solids (VS) of FW and BS were determined according to the previous analytical method (APHA, 2005). After recording the weights of FW and BS, the samples were dried at 105 °C for 24 h. After 24 h, FW and PS were weighed again to determine the TS content as shown in Equation 1. In the second step, the dried samples were heated in a muffle furnace to 550 °C for two hours to turn them into ash. The samples were taken out of the furnace, cooled to room temperature, and then weighed again. The VS content was calculated as shown in Equation 2.

[0048] where m1 is the weight of the empty crucible, m2 is the weight of the crucible and the raw material before drying, m3 is the weight of the crucible and the raw material after drying, and m4 is the weight of the crucible and the ash after the muffle furnace.

[0049] Assume that the total of C, H, O, and N in volatile solids (VS) accounts for 99.5%. The contents of C, H, N, and O are quantitatively analyzed using an elemental analyzer, and the data of C, H, O, and N in the volatile solids are used to determine the contents of C, H, N, and O in food waste. The pH value is measured using an automatic pH meter (AquaSearcherTM, OHAUS, China). COD and ammonia nitrogen are determined using a HACH kit (HACH Company, USA), and the operation method is carried out according to the kit instructions. High-performance liquid chromatography (HPLC) (Agilent) equipped with an ICSep ICE-Coregel 87H3 column (USA) is used for lactic acid determination and VFA analysis. The mobile phase is a 5 mM H2SO4 solution, the flow rate is 0.6 mL / min, and the pressure is 60 kPa.

[0050] 1.4 Anaerobic digestion (AD) Anaerobic digestion is repeated under aerobic and anaerobic pretreatment conditions. The ratio of raw material to inoculum is 1:10 (v / v), and FW pretreated aerobically and anaerobically is inoculated with concentrated BS, with a working volume of 0.8 mL. To ensure anaerobic conditions, high-purity nitrogen is injected into the anaerobic bottle, sealed, and cultured in a shaker at 37 °C for 8 days, with a shaker speed of 130 rpm. Samples are collected daily to analyze the concentrations of VFA and lactic acid. Figure 1 shows a schematic diagram of the experimental setup.

[0051] 1.5 Strains, media, and culture conditions Escherichia coli strains are grown in Luria–Bertani medium containing 50 mg / L kanamycin, and the culture conditions are 37 °C and 180 rpm.

[0052] Used in the present invention Schizochytrium limacinumThe SR21 strain is stored in this laboratory in the form of a frozen culture. The cells stored in 50% (v / v) glycerol at -80 °C are cultured in two stages, namely the seed culture stage and the fermentation culture stage. The seed medium contains glucose (30 g / L), yeast extract (8 g / L), and artificial seawater crystal (20 g / L). The fermentation medium consists 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 fermentation broth of FW is centrifuged to obtain the fermentation broth, and then the impurities in the fermentation broth are filtered using filter paper. The components of the fermentation medium are added to the filtered fermentation broth, and the VFA in the fermentation broth is used as a carbon source for the cultivation of Schizochytrium, with the cultivation temperature at 28 °C and the shaker speed at 180 rpm. The VFA fermentation medium under different initial pH conditions is obtained by sterilizing the treated VFA fermentation medium and then adjusting the pH of the fermentation broth to 5, 6, 7, 8, and 9 respectively using 5 M NaOH. The pH value of the medium is detected using a portable pH meter, and the remaining cultivation conditions remain unchanged. The media with different VFA concentrations are 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 fermentation broth as carbon sources, and then other components of the fermentation medium are added. After stirring evenly, they are sterilized, and then the pH is adjusted using 5 M NaOH. All media are sterilized at 115 °C for 20 minutes.

[0053] 1.6 Plasmid construction Escherichia coli DH5α is used for plasmid construction and plasmid enrichment. By comparing the annotated genes in NCBI, the genes for lactate dehydrogenase (d-LDH, l-LDH) were predicted from the Schizochytrium limacinum SR21 genome. The d-LDH and l-LDH genes were amplified from the Schizochytrium genomic DNA using the corresponding primers, and each gene was inserted into the pK2-PA-(ApaI / KpnI)cyc-PE-NPTII vector digested with ApaI and KpnI. The lactate oxidase (LOX) gene from Aerococcus viridans was obtained after codon optimization, and the amplified SpLOX fragment was also inserted into the pK2-PA-(ApaI / KpnI)cyc-PE-NPTII vector digested with ApaI and KpnI. In this experiment, the synthesis of PCR primers, sequencing, and gene synthesis services were all completed by Genewiz (Suzhou) Co., Ltd. The specific primers are shown in Attachment Table 1.

[0054] Table 1. Primers used in the present invention 1.7 Transformation of Schizochytrium The constructed vector was transformed into Schizochytrium using the particle bombardment method. First, 30 mL of Schizochytrium sp. culture was centrifuged at 6000 r / min for 5 min, washed twice with 20 mL of sterile water, and the waste liquid was discarded. The cells were resuspended in 1 mL of sterile water, and 200 μL of the cell suspension was spread on a solid seed plate and air-dried for fixation. Preparation of microparticles: 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. Then the mixture was washed twice with 200 μL of absolute ethanol and centrifuged at 6000 r / min to obtain the DNA-coated gold microparticle carrier. Next, the gold microparticle carrier was resuspended in 70 μL of ethanol as the bullet for particle bombardment. The solution containing the DNA-coated gold microparticle carrier was spread on the microparticle carrier disk, and the microparticle carrier was bombarded into the cells using the Biolistic PDS-1000 / He system (Bio-Rad, USA) with the parameter settings of 1100 psi pressure and 6 cm target distance. After bombardment, the plate was incubated at 28°C for 16 - 24 hours. Then, the cells were spread on a solid seed plate containing 150 mg / L G418 and incubated at 28°C for 5 - 7 days to screen for transformants.

[0055] The single colonies on the resistant plate were stained using a GUS staining kit (Coolaber, SL7160) for preliminary screening of transformants. The obtained transformants were subcultured 5 times and further verified by PCR amplification of the target gene.

[0056] 1.8 Determination of Biomass and Protein Content We used the method of dry cell weight (DCW) to evaluate the biomass as described in a previously published study. After the cultivation was completed, the same volume (V) of the cell suspension was placed into a 50 mL pre-weighed centrifuge tube (M1) and centrifuged at 6000 r / min for 10 minutes at 4°C. The cells were washed three times with distilled water, and the cells were placed in a -20°C ultra-low temperature freezer and frozen overnight. Then the cells were freeze-dried using a freeze dryer (FD-1C-50, BJBYK Company, China), and the weight of the centrifuge tube and the cells was weighed and recorded as M2. The dry cell weight was calculated using Equation 3.

[0057] Similarly, the protein content was measured using the research method published by the laboratory before. The freeze-dried bacterial cells were ground into powder, and an appropriate amount of powder (3 - 4 mg) was weighed. The nitrogen content (%) was measured using a Flashsmart elemental analyzer (Thermo Ltd., Waltham). The protein content (%) was calculated using calculation formula 4.

[0058] 1.9 Determination of lipid content and fatty acid composition analysis The improved method from our previous early research was used to determine the total lipid content. Using a gas chromatograph (GC-2010, Shimadzu, Japan), the fatty acids in the lipid were converted into fatty acid methyl esters (FAME). The temperature of the gas chromatograph was initially set at 180 °C and then increased to 240 °C at a rate of 30 °C per minute within 18 minutes. The temperatures of 250 °C and 260 °C were selected as the injection port and the flame ionization port, respectively. The injection volume was 1 μL, the split ratio was 30:1, and nitrogen was used as the carrier gas.

[0059] 1.10 Statistical analysis 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 the significant differences in the experimental data. A P-value less than 0.05 was considered statistically significant.

[0060] 2 Results and discussion 2.1 Characteristics of food waste and inoculum Table 2 lists the characteristics of the FW and inoculum used in the study. The TS (35.3%) and VS (10.5%) of FW are higher than those of the inoculum (TS: 1.6%, VS: 1.5%). The VS / TS ratio of FW is 29.6%, while the VS / TS ratio of the inoculum is significantly higher, at 94.6%. The initial pH value of FW is 5.3, and the initial pH value of the inoculum is 8.0. The pH values were adjusted to 7.0. The nitrogen content (49.3%) of FW is higher than that of the inoculum (3.6%), while the carbon content of the inoculum is 25.8% higher than that of FW (4.0%). In addition, the hydrogen content (6.8%) and oxygen content (37.2%) of FW are higher than those of the inoculum. The C / N ratio of FW is 12.3, and the C / N ratio of the inoculum is 7.2, indicating that a balanced nutrient composition is beneficial for the FW fermentation inoculum to produce VFA through AD.

[0061] Table 2. Characteristics of food waste and inoculum Let A represent the total weight of the sample, and the sample, and B represent TS 2.2 VFA production under conditions of micro-aeration pretreatment and different OL conditions To better understand the degradation of FW and the production of target compounds, the present invention conducted batch fermentation experiments, focusing on how FW produces VFA through fermentation. The results elucidated the complex relationship between microbial interactions and metabolic processes during VFA synthesis. In particular, the present invention examined the VFA composition and synthesis under different OL conditions (5, 10, 15, 20, and 25 gVS / L) with and without micro-aeration and heat pretreatment conditions, thereby providing an in-depth understanding of the impact of these pretreatment technologies on the process.

[0062] 2.2.1. Production of lactic acid As shown in Figure 2, with or without micro-aeration pretreatment, as the organic loading rate (OLR) increased from 5 gVS / L to 25 gVS / L, the lactic acid production increased significantly with the increase in OLR and the presence of oxygen. At the lowest OL of 5 gVS / L, under anaerobic conditions, the lactic acid production increased slightly from 2.1 g / L to 10.3 g / L within 8 days, and increased to 11.1 g / L under aerobic conditions. In addition, at OL of 10 gVS / L and 15 gVS / L, the increase in lactic acid production due to oxygen pretreatment became more obvious, reaching 17.3 g / L and 21.3 g / L respectively. At an OLR of 20 gVS / L, a significant increase in lactic acid production was observed under aerobic conditions to 26.9 g / L, while it was 22.8 g / L under anaerobic conditions. Interestingly, under the condition of the highest OL of 25 gVS / L, the lactic acid production decreased slightly, reaching 18.9 g / L under anaerobic conditions, while increasing to 19.6 g / L under aerobic conditions. 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 continuously enhanced lactic acid production in all OLs, indicating its key role in improving fermentation efficiency and potentially contributing to the breakdown of complex substrates into more fermentable sugars for lactic acid bacteria (LAB). The mechanism of lactic acid production involves the anaerobic fermentation of carbohydrates by LAB. Initially, carbohydrates are decomposed into glucose and then converted to pyruvate through glycolysis. Subsequently, pyruvate is reduced to lactic acid by lactate dehydrogenase, a process that regenerates NAD from NADH +, thus enabling glycolysis to continue. This biochemical pathway not only emphasizes the importance of optimal conditions for LAB activity but also illustrates the key role played by oxygen in potentially enhancing the initial breakdown of complex carbohydrates, making the substrate more fermentable. These findings highlight the importance of balancing OLR and oxygen pretreatment to optimize lactic acid production, which is a key parameter in industrial fermentation processes aimed at achieving efficient lactic acid production.

[0063] 2.2.2. Acetic acid production Figure 3 shows the effect of OL level and the presence of oxygen on acetic acid production. At the lowest OL of 5 gVS / L, acetic acid production was extremely low, but it increased slightly in the presence of oxygen. As the OL level increased, the production under anaerobic conditions was always lower than that under aerobic conditions. This trend was evident at all OL levels, with the most significant increase observed at higher OLs (20 gVS / L and 25 gVS / L), where the production under anaerobic conditions was lower than that under aerobic conditions, being 3.4 g / L and 4.0 g / L respectively, while the production under aerobic conditions was 4.5 g / L and 4.4 g / L respectively. These results clearly show that introducing oxygen into this anaerobic process can improve the efficiency of AD by selectively inhibiting or promoting certain microbial activities. Oxygen can inhibit methanogenic archaea as they are sensitive to oxygen, thus reducing their competition with acidogenic bacteria for the substrate. This will lead to an increase in the production of volatile fatty acids (including acetic acid) as acidogenic bacteria can access more substrate. However, excessive oxygen will result in complete aerobic digestion, thus reducing acetic acid production as acetic acid will be further decomposed into carbon dioxide and water. The production of acetic acid 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 hexose to acetic acid in the absence of oxygen. The presence of oxygen not only affects the choice of metabolic pathway but also increases the total production of acetic acid. Therefore, appropriate micro-aeration treatment is required for acetic acid production.

[0064] 2.2.3. Butyric acid production Butyric acid production varied with different OL levels, and significant differences were observed when comparing aerobic and anaerobic conditions, as shown in Figure 4. For example, at 10 gVS / L OL, the presence of oxygen increased the butyric acid concentration from a lower value of 5.6 g / L observed under anaerobic conditions to 5.8 g / L. This trend was more pronounced at 15 gVS / L OL, where the butyric acid concentration reached a maximum of 6.7 g / L under aerobic conditions, significantly higher than 5.1 g / L observed under anaerobic conditions. In contrast, at the highest OL of 25 gVS / L, the concentration peak was much lower, with a maximum concentration of only 1.7 g / L under anaerobic conditions and 0.7 g / L under aerobic conditions, indicating that very high substrate concentrations or other limiting factors may have an inhibitory effect at this OL level. The reasons for these results may be due to the anaerobic fermentation process carried out by the genus Clostridium, which metabolizes carbohydrates to butyric acid and other volatile fatty acids through the acetyl-CoA pathway. In some cases, the introduction of oxygen seems to enhance butyric acid production, which may be due to the activity of aerobic or facultative anaerobic bacteria that can utilize alternative metabolic pathways to produce butyric acid. For example, at a lower OL level (5 gVS / L), the introduction of oxygen slightly increased the maximum butyric acid concentration from 3.4 g / L to 3.8 g / L, indicating that oxygen can affect microbial kinetics and fermentation results. Considering the complex interactions between microbial metabolism, substrate availability, and environmental conditions, these results emphasize the importance of balancing OL levels and oxygen availability for optimizing butyric acid production.

[0065] 2.2.4. Formic acid production The production of formic acid revealed the effects of OL concentration and oxygen supply on the food waste degradation process. As Figure 5 shown, at all OL levels, the presence of oxygen consistently led to a decrease in formic acid concentration compared to anaerobic conditions. At the lowest OL of 5 gVS / L, the formic acid concentration on the 8th day ranged from 0.1 g / L under anaerobic conditions to 0.09 g / L under aerobic conditions. Similarly, at the highest OL of 25 gVS / L, the concentration range on the 8th day was from 0.1 g / L under anaerobic conditions to 0.06 g / L under aerobic conditions. However, at higher OL levels (15 gVS / L and 25 gVS / L), the effect of oxygen seemed to weaken, indicating that there may be a saturation effect or alternative degradation pathways in the case of an increase in the initial formic acid concentration. The interaction between the initial formic acid concentration, oxygen availability, and degradation rate highlights the complex dynamics of the possible formic acid degradation mechanism of certain microorganisms during the food waste degradation process, and further comprehensive studies are needed to clarify the potential chemical pathways.

[0066] 2.2.5. Propionic acid production Figure 6 shows that under different initial concentrations and conditions (anaerobic and aerobic), the propionate level increased in a concentration-dependent manner over 8 days. When OL was 25 gVS / L, the highest value was 1.4 g / L under anaerobic treatment and 1.3 g / L under aerobic treatment. Conversely, the lowest value occurred at 5 gVS / L on day 1, with a propionate concentration of 0.10 g / L under anaerobic treatment conditions and 0.12 g / L under aerobic treatment conditions. Compared with non-oxidizing conditions, the presence of an oxidant seemed to slightly increase the propionate level, indicating a potential stabilizing or enhancing effect. Overall, at all initial concentrations, the propionate concentration increased with time under both conditions, suggesting that the accumulation of propionate is time-related. In microorganisms, the degradation of propionate may involve multiple mechanistic pathways. In the presence of oxygen, one possible pathway is the oxidation of propionate to form propionyl-CoA, which can further undergo oxidative decarboxylation to generate acetyl-CoA and succinate. Acetyl-CoA can then enter the TCA cycle to produce energy. The presence of an oxidant may promote these oxidation reactions, resulting in the observed increase in propionate levels. In the absence of an oxidant, alternative pathways such as fermentation or anaerobic metabolism may dominate, potentially producing different intermediate products and metabolic outcomes.

[0067] 2.2.6. Total VFA production Figure 7 shows the dynamic curves of total volatile fatty acid (VFA) production under different OL conditions, with particular attention paid to 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 research results provide several key insights into the changing trends and yields of VFA production in these controlled environments. Starting from the lowest concentration of 5 gVS / L, the total VFA gradually increased from 3.4 g / L to 14.2 g / L under anaerobic conditions. In contrast, the presence of oxygen promoted an increase in VFA concentration, with the total VFA concentration increasing from 4.0 g / L to 16.1 g / L, indicating that oxygen treatment may catalyze or facilitate the more efficient conversion of food waste into VFA. When the OL was 10 gVS / L, a similar trend was observed, but the overall VFA production was higher. In the absence of oxygen, the final VFA concentration was 22.2 g / L. Under aerobic treatment, the highest value reached 24.5 g / L on the 8th day. This pattern emphasizes the role of oxygen treatment in enhancing VFA production, and this trend becomes more obvious at higher substrate concentrations. At an OL of 15 gVS / L, the VFA concentration under anaerobic conditions (26.4 g / L) was significantly lower than that under aerobic conditions (30.0 g / L), which further confirmed that oxygen treatment not only promotes VFA production, but its effect is amplified at higher substrate concentrations. The 20 gVS / L concentration presented an interesting anomaly, especially under oxygen treatment conditions, where the VFA was initially 10.2 g / L and increased sharply to 37.0 g / L on the 8th day. This indicates that there is a threshold or critical point during the anaerobic fermentation process, at which the combined action of oxygen and substrate concentration amplifies VFA production. Finally, under the condition of OL being 25 gVS / L, the data showed a plateau effect, with the VFA without oxygen treatment slightly decreasing from the peak, increasing from 9.6 g / L on the 1st day to 24.8 g / L on the 8th day, while after oxygen treatment, the VFA concentration increased from 10.1 g / L to 25.8 g / L. This indicates that there is a potential saturation point or limit to the catalytic effect of oxygen at high substrate concentrations. These results clarify that there is an obvious dependence relationship between VFA production and OL concentration, and the presence of oxygen has a significant impact on it. Oxygen treatment can continuously increase VFA production at all OL concentrations, and its effect becomes more obvious at medium concentrations, but may reach a saturation point at an OL of 25 gVS / L. This intricate interaction between substrate concentration, the presence of a small amount of oxygen, and VFA production highlights the complex biochemical mechanisms involved, which may involve enhancing oxidation pathways or improving substrate utilization in the presence of oxygen.

[0068] 2.3 Ammonia nitrogen and COD concentrations under different organic loads during anaerobic digestion of food waste under aerobic and anaerobic conditions Figure 8 shows data on ammonium nitrogen concentrations monitored under aerobic and anaerobic conditions at different OL concentrations (5 gVS / L, 10 gVS / L, 15 gVS / L, 20 gVS / L, and 25 gVS / L), revealing the complex interactions of biological and chemical processes influencing nitrogen dynamics in these systems. At a concentration of 5 gVS / L, the presence of oxygen seemed to slightly reduce the ammonium nitrogen concentration 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, thereby reducing ammonia levels. When the substrate concentration increased to 10 gVS / L and 15 gVS / L, we observed an overall upward trend in ammonium nitrogen concentration, especially 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 concentration, especially under aerobic conditions, may reflect the dynamic balance between ammonification and nitrification processes. At OLs of 20 gVS / L and 25 gVS / L, the ammonium nitrogen concentration reached its peak, with the highest concentrations observed on day 8 under anaerobic conditions being 0.45 g / L and 0.50 g / L, respectively. These elevated levels may indicate saturation or inhibition of the nitrification process, possibly due to the inhibition of the ability of nitrifying bacteria by the higher organic load or the accumulation of inhibitory substances that impede bacterial activity.

[0069] As shown in Figure 9, the experimental results of COD at different monitored substrate concentrations provide profound insights into the oxidation behavior of organic substances in the sample. Under the condition of 5 gVS / L, without oxygen, the COD value gradually increases from 18.6 g / L to 28.2 g / L. In the presence of oxygen, the COD concentration reaches from 18.3 g / L to 26.7 g / L, which is lower than that without oxygen. This indicates that oxygen has little effect on the oxidation process at a lower OL concentration. At a concentration of 10 gVS / L, the increase in the COD value without oxygen is more obvious, increasing from 33.0 g / L on the first day to 47.3 g / L. On the contrary, in the presence of oxygen, it reaches from 18.4 g / L to 47.1 g / L, almost the same as the COD concentration without oxygen. Oxygen can significantly accelerate the oxidation process, and the results at an OL concentration of 15 gVS / L confirm this conclusion. Without oxygen, the COD value increases to 33.5 g / L and then to 71.28 g / L on the 8th day. With oxygen, it significantly increases from 29.5 g / L to 71.9 g / L. At a concentration of 20 gVS / L, the COD concentrations without oxygen and with oxygen are almost the same. Starting from 36.6 g / L without oxygen, the highest value reaches 104.8 g / L, indicating a relatively large organic load. With oxygen, the initial value is also 36.5 g / L, but the final value is slightly lower, at 104.4 g / L. The most significant increase in COD is observed at the highest OL concentration of 25 gVS / L. Without oxygen, the COD value starts from 43.0 g / L and increases to 109.2 g / L on the 8th day. With oxygen, the initial value is 40.8 g / L and the peak value is 111.8 g / L, indicating that the effect of oxygen on the oxidation of organic compounds is more obvious at higher concentrations. And previous studies have shown that these observations highlight an obvious trend: the COD value increases with the increase in substrate concentration, indicating a higher organic load and a greater demand for oxygen for effective oxidation. The presence of oxygen usually increases COD, which indicates that oxygen helps to more thoroughly oxidize organic matter, emphasizing its importance in environmental and wastewater management strategies aimed at reducing the impact of organic pollutants.

[0070] 2.4 Schizochytrium limacinum SR21 uses VFA to produce oil-rich single-cell protein 2.4.1 Effects of different initial pH conditions on the utilization of VFA by Schizochytrium The pH value is one of the important influencing factors affecting microbial growth and metabolite synthesis. On the one hand, pH can change the charge of the protoplasmic membrane inside the cell, thereby affecting the absorption of nutrients by microorganisms and further affecting the growth of the bacterial cells. On the other hand, the activity of enzymes in microorganisms is affected by pH, thereby affecting the metabolic regulation in the organisms and further affecting the synthesis of products. In order to find the pH value suitable for the growth of bacterial cells and product synthesis, Schizochytrium limacinum The initial pH fermentation optimization results of SR21 in VFA fermentation culture provide valuable insights into the interactions between changes in pH levels, VFA consumption, and microbial growth.

[0071] The data presents a systematic evaluation of lactic acid and VFA concentrations (including formic acid, acetic acid, propionic acid, and butyric acid) under different conditions with initial pH values ranging from 6 to 9. The utilization of lactic acid and volatile fatty acids by Schizochytrium sp. SR21 under different initial pH conditions was studied in detail, thus providing in-depth understanding of the metabolic preferences and efficiencies of Schizochytrium sp. As shown in Figure 10, this study indicates that the initial pH conditions of 6 - 8 are favorable for the consumption of organic acids, highlighting the optimal environment for acid metabolism. Within 0 - 48 h, the contents of formic acid, acetic acid, propionic acid, and butyric acid showed a continuous decreasing trend, and within 72 h, these acids were all consumed. The consumption of lactic acid was just the opposite. In the early stage of 0 - 48 h, due to the consumption of other acids, there was a certain inhibitory effect on the bacterial cells' intake of lactic acid, resulting in less consumption of lactic acid. After 72 h, the content of lactic acid continued to decrease, but there was still some lactic acid residue in the fermentation broth after 144 h of fermentation. The consumption of organic acids at an initial pH value of 7 indicates that neutral or slightly acidic conditions may enhance the activity of enzymes involved in decomposing these acids. Interestingly, at an initial pH value of 5, there was almost no change in the pH of the culture broth throughout the fermentation process ( Figure 11 as shown in a) below). This may be due to the fact that the cells were stressed by acid, and cell growth was completely inhibited or even died, resulting in the VFA in the fermentation broth not being utilized, so the pH of the fermentation broth remained unchanged all the time. At an initial pH value of 9, a decrease in the pH value of the fermentation broth was detected within the first 24 h. It is speculated that this may be because the cells excreted acidic small molecules and carbon dioxide in the cells in order to adapt to the environment, resulting in a decrease in the pH value of the fermentation broth ( Figure 11 as shown in a) below). And 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 10In d), this is exactly the same as the change in pH value in the fermentation broth. This indicates that during the early stage of organic acid utilization, in order to resist the harsh external environment, cell growth is inhibited and there is a growth stagnation period. In addition, when the initial pH is 6 and 7, the highest biomass and protein production can reach 16.42 g / L and 6.13 g / L respectively, which shows that pH 6 and 7 are the optimal conditions for acid utilization ( Figure 11 In b). Similarly, previous studies have investigated the effects of culture conditions, pH, carbon sources, and nitrogen sources on Schizochytrium sp. S31 and found that when the initial pH is 7, Schizochytrium sp. S31 can accumulate up to 40% biomass and lipids with a DHA content of 13% (w / w). However, when detecting the oil content and fatty acid composition in cells, it was found that the oil content increased significantly under the initial pH 9 condition, reaching up to 21.58%, but the content of unsaturated fatty acids mainly composed of DHA decreased, while the content of saturated fatty acids mainly composed of C16:0 increased ( Figure 11 In c). This indicates that there may be a trade-off or optimization requirement between achieving metabolic efficiency and supporting cell growth, which may have an impact on high-density growth of microbial cells and efficient substrate utilization.

[0072] Throughout the study, the consumption trend of VFA indicates that the pH optimization strategy can enhance metabolic activity and biomass production in the culture. It is very obvious that the performance in terms of acid utilization and cell growth is poor under the initial pH 9 condition. This pH level has a significant inhibitory effect on cell growth, highlighting the importance of precise pH control in the microbial culture environment. The initial pH 7 is considered to be beneficial to the metabolic process of Schizochytrium sp., and its preference for slightly acidic conditions enables maximum growth, which provides valuable insights for biotechnological applications. Overall, this study emphasizes the key role of pH in influencing microbial metabolism and growth dynamics. This pH balance helps to effectively decompose acids and support cell growth, highlighting the importance of controlled environmental conditions in industrial biological applications.

[0073] 2.4.2 Effects of Different VFA Concentrations on the Growth of Schizochytrium sp. SR21 To further study the utilization of VFA by Schizochytrium sp., the experiment monitored the utilization of lactic acid, formic acid, acetic acid, propionic acid, and butyric acid by Schizochytrium sp. at different initial concentrations (25%, 50%, 75%, and 100% VFA), as shown in Figure 12. The utilization of VFA by Schizochytrium sp. SR21 provides valuable insights into the metabolic capabilities and preferences of this microorganism, and at the same time reveals how microbial strains adapt to and metabolize different VFAs.

[0074] Under different concentrations of VFA, the utilization of lactic acid, formic acid, acetic acid, propionic acid, and butyric acid by Schizochytrium is different. Under 25% VFA and 50% VFA conditions, after 48 h of fermentation, except for lactic acid, the other acids were all consumed. This indicates that when the substrate concentration is low, cell catabolism can be better utilized to support the rapid growth of microorganisms. Under 75% VFA and 100% VFA conditions, formic acid, propionic acid, and butyric acid were all consumed within 48 h, while acetic acid was consumed within 144 h of fermentation. In contrast, the degradation rate of lactic acid is slower, and some acid remained unconsumed after the fermentation ended, indicating that the enzyme was under high-concentration lactic acid stress, with a lower affinity for the substrate, or more complex and energy-consuming pathways were required to promote their decomposition. According to existing research, high substrate concentrations can inhibit the activities of important enzymes such as glycerol kinase and glycerol-3-phosphate dehydrogenase. Therefore, in subsequent experiments, we need to further optimize and enhance the microbial degradation of lactic acid. In addition, during the fermentation process, the pH value of the fermentation broth increased rapidly in the early stage and slowly in the later stage, and finally reached about 9.2 ( Figure 13 in a). This change is related to the degradation of VFA, indicating that the strain rapidly utilized these acids as a carbon flux and energy source in the early stage. The increase in pH value over time further supports the hypothesis that acid degradation occurs, resulting in a decrease in environmental acidity. In addition, Figure 13 b in shows the biomass and protein content under different VFA concentrations. Interestingly, as the VFA concentration increased, the biomass of Schizochytrium gradually increased, but the protein content gradually decreased. Under 25% VFA conditions, the protein content of Schizochytrium could reach 49.35%, which was 11.22% higher than that under 100% VFA conditions; under 100% VFA conditions, the biomass and protein production of Schizochytrium increased significantly, reaching a maximum of 15.29 g / L and 5.83 g / L respectively, which were 121.59% and 71.47% higher than those under 25% VFA conditions. It is speculated that the increase in the carbon-nitrogen ratio may lead to a decrease in the total protein content. Similarly, existing research has shown that when studying the conversion of rice hydrolysate (BRH) into protein using microalgae, it was found that a low C / N ratio is beneficial to the synthesis of protein in microalgae cells. At the same time, the oil content, fatty acid composition, and content under different concentrations were also detected, but there were no significant changes ( Figure 13 in c).

[0075] Therefore, in this study, although a higher concentration of VFA is beneficial for biomass production, an increase in the carbon-nitrogen ratio will affect the accumulation of microbial single-cell content. This insight is crucial for scaling up this process, as it indicates the need to precisely control the VFA concentration to avoid inhibitory effects on microbial metabolism. In summary, in subsequent experiments, we will select a fermentation medium with pH 7 and 100% VFA for the cultivation of Schizochytrium.

[0076] 2.4.3 Expression of lactate dehydrogenase / lactate oxidase genes in Schizochytrium improves the utilization of lactate In previous studies, it was found that when Schizochytrium utilized VFA, lactate was not fully utilized, indicating that the lactate dehydrogenase activity in Schizochytrium was low, or the lactate concentration was too high, causing stress to Schizochytrium, resulting in a low utilization rate of lactate. Genes related to lactate utilization have been widely studied in various microorganisms. The enzymes encoded by these genes are mainly involved in the lactate metabolic pathway, including the oxidation, dehydrogenation of lactate, and the further conversion into other metabolites. Among them, the lactate dehydrogenase (LDH) gene is the most common core gene in lactate metabolism, responsible for catalyzing the reversible conversion between lactate and pyruvate. LDH can be divided into two subfamilies (d-LDH and l-LDH) according to the different substrates. In addition, the enzyme encoded by the lactate oxidase (LOX) gene is a flavin mononucleotide (FMN)-dependent flavoenzyme, which can directly oxidize lactate to pyruvate in the presence of oxygen (O2) without the need for the cofactor NAD + to be present ( Figure 14 ). Since LOX binds tightly to its cofactor FMN, it does not require additional supplementation of FMN during biotransformation, thus having great potential and application value in industrial applications. LOX has been found in various microorganisms, including Pediococcus sp., Aerococcus viridans , Streptococcus iniae , Geotrichum candidum and Lactococcus lactis . Among various LOXs, the LOX from A. viridans (AvLOX) has the highest activity towards lactate and has been intensively studied for use in biosensors to measure lactate concentration in blood or other body fluids.

[0077] To enable Schizochytrium to better utilize the by-product lactate generated from food waste, the present invention expressed the endogenous LDH and the LOX from Aerococcus viridans ( A. viridans) LOX to enhance the utilization of lactic acid by Schizochytrium. The d-LDH, l-LDH, and SpLOX genes were expressed in cells using the constitutive promoter PA. The pK2-PE-NPTII vector only contained the resistance gene and the GUS gene, which were 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. The recombinant strains d-LDH, l-LDH, and PE amplified the GUS-NPTII gene fragment (2661 bp) by PCR, while the recombinant strain spLOX-2 successfully amplified the SpLOX gene fragment (1122 bp) by PCR ( Figure 15 b). To evaluate the ability of the overexpressing strains to utilize lactic acid, the recombinant strains (d-LDH, l-LDH, SpLOX, PE) and the wild-type SR21 were fermented and cultured in the VFAs fermentation medium. Figure 16 shows the consumption of VFAs by 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 h. Surprisingly, after fermentation, it was found that the residual lactic acid content in the recombinant strain spLOX-2 was the lowest, 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, improving the utilization of lactic acid. At the same time, due to the increased utilization of lactic acid by the recombinant strain spLOX-2, its biomass was also significantly higher than that of the wild-type strain, reaching up to 17.46 g / L ( Figure 17 c), which was significantly increased by 14.16% compared to the wild-type. Moreover, 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 strain ( Figure 17 d). It is speculated that lactic acid is decomposed into pyruvate under the action of lactate oxidase, and the increased utilization of lactic acid leads to an increase in pyruvate content. The main destinations of pyruvate in microorganisms are nitrogen metabolism, amino acid metabolism, and conversion into acetyl-CoA under the action of propionate dehydrogenase to enter the fatty acid synthesis pathway or decomposition into oxaloacetate by pyruvate carboxylase to enter the TCA cycle ( Figure 14)。Schizochytrium is an oil-producing microorganism with strong fatty acid metabolism. The increase in pyruvate content is very likely to increase the content of acetyl-CoA. Acetyl-CoA is an important precursor in the oil synthesis pathway. The increase in acetyl-CoA will upregulate the oil synthesis pathway, thereby increasing the oil content of the recombinant strain spLOX-2, but the specific mechanism still needs further research.

[0078] At the same time, Schizochytrium spLOX-2 was deposited. The taxonomic name of the Schizochytrium spLOX-2 strain is: Schizochytrium Schizochytrium sp. , the strain Schizochytrium Schizochytrium sp. spLOX-2 was deposited in the China General Microbiological Culture Collection Center (abbreviated as CGMCC), with the deposit number: CGMCC No. 41623, the deposit time: November 11, 2024, and the address of the deposit unit: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0079] Therefore, the expression of lactate oxidase in Schizochytrium improves the utilization of lactate by Schizochytrium, enabling Schizochytrium to efficiently convert VFA from food waste into oil-rich single-cell protein, providing a new path for the sustainable biorecycling economy model. However, since the process of treating food waste is relatively complex, further research will focus on scaling up the process and improving the economic feasibility of this technology in industrial applications. In summary, these research results lay the foundation for further exploring Schizochytrium limacinum the mechanism of SR21's utilization of VFA and are of great significance for biotechnological applications such as the production of value-added chemicals and the microbial fermentation process.

[0080] 3 Conclusions In the present invention, the innovative combination of micro-aeration and thermal pretreatment significantly enhances the enzymatic hydrolysis of food waste, thereby increasing the production of VFAs. Meanwhile, through fermentation optimization, the crucial role of pH in maximizing the utilization of VFAs by Schizochytrium and microbial growth is discovered, where an initial pH of 7 is considered beneficial for the metabolic process of Schizochytrium. This pH balance helps to effectively decompose acids and support cell growth, highlighting the importance of controlled environmental conditions in industrial biological applications. In addition, the research shows that while higher concentrations of VFAs are beneficial for biomass production, an increase in the carbon-nitrogen ratio affects the accumulation of microbial single-cell content. This insight is crucial for scaling up the process as it indicates the need for precise control of VFA concentration to avoid inhibitory effects on microbial metabolism. Also, through the expression of lactate oxidase in Schizochytrium, the utilization of lactate is significantly improved, resulting in a significant increase in the biomass of Schizochytrium and the production of single-cell protein rich in high-value oils. This article demonstrates the great potential of Schizochytrium for the utilization of VFAs from food waste. Therefore, this research provides a good foundation for the development of a sustainable bioeconomic model in which food waste is not only disposed of but also converted into economically valuable products. This not only helps to reduce environmental pollution but also contributes to the reuse of resources, in line with global sustainable development goals. Future research will aim to improve and scale up the process and explore its commercial feasibility on a larger scale.

[0081] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the embodiments of the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the embodiments of the present invention are not limited to the specific details and the embodiments shown and described herein.

Claims

1. A Schizochytrium strain with high efficiency in converting food waste into single cell protein, characterized in that, The Schizochytrium strain is Schizochytrium spLOX-2, and the taxonomic naming of the Schizochytrium spLOX-2 strain is: Schizochytrium Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. spLOX-2 is deposited in the China General Microbiological Culture Collection Center (abbreviated as CGMCC), with the deposit number: CGMCC No. 41623, the deposit date: November 11, 2024, and the address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 2. Use of the Schizochytrium sp. strain according to claim 1 in food waste treatment and single cell protein production.

3. A method for efficiently converting kitchen waste into single-cell protein, characterized in that, Comprising the following steps: 1) Ferment food waste to produce volatile fatty acids through microaeration and heat pretreatment; 2) Use the volatile fatty acids produced in step 1) as a carbon source to culture Schizochytrium sp. After culturing for several days, obtain single cell protein from the cultured cells.

4. The method for efficiently converting kitchen waste into single-cell protein according to claim 3, characterized in that, In step 1), fermenting food waste to produce volatile fatty acids comprises the following steps: Take the food waste, dry it to make food waste powder, then take the food waste powder, the original inoculum seed liquid and water and place them in a sealed container, remove air to form an anaerobic environment, and then inject oxygen into the sealed container, and carry out anaerobic digestion for 1 - 8 days under microaeration conditions to obtain the fermented product of food waste, and harvest the fermentation broth.

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

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

7. The method for efficiently converting kitchen waste into single-cell protein according to claim 3, characterized in that, In step 2), the method of using the volatile fatty acids produced in step 1) as a carbon source is to add a fermentation medium for Schizochytrium sp. to the fermentation broth of food waste fermentation in step 1), adjust the initial pH to 6 - 9 to form a final fermentation medium, and culture Schizochytrium sp. with the final fermentation medium. The concentration of volatile fatty acids in the final fermentation medium is: 13 - 45 g / L.

8. The method for efficiently converting kitchen waste into single cell protein according to claim 7, characterized in that, The fermentation medium for Schizochytrium sp. contains components with the following concentrations: 5 g / L of yeast extract, 0.3 g / L of NaCl, 1 g / L of K2SO4, 0.1 g / L of KH2PO4, 4 g / L of MgSO4·7H2O, 0.05 g / L of CaCl2.

9. The method for efficiently converting kitchen waste into single-cell protein according to claim 4, characterized in that, In step 1), the ratio of food waste powder to the original inoculum seed liquid is 1:10 (v / v).

10. The method for efficiently converting kitchen waste into single-cell protein according to claim 3, characterized in that, The Schizochytrium sp. used is the wild - type Schizochytrium sp. SR21.

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