Method for recycling waste liquid in production of venus armeria protein

Abstract

The application discloses a method for recycling waste liquid in the production process of Venetian Fusarium protein, which comprises the steps of component analysis, nutrient supplement, fermentation verification and recycling evaluation, and aims at the problems of high water consumption and high emission in the preparation process of microbial protein of the filamentous fungus Venetian Fusarium, and builds component analysis, resource utilization potential evaluation and recycling process demonstration of two types of typical process waste liquid. Through the hierarchical recycling strategy design of setting the fermenting waste liquid to be reused once and the de-nucleic acid waste liquid to be reused twice, the theoretical production amount of the two kinds of waste water is reduced from 18.8 t to 7.7 t under the condition of four production cycles, and the emission reduction ratio is about 59.04%, which shows that the method has significant water saving and emission reduction potential in theory, and can provide data support and technical basis for the optimization of the green manufacturing process of the filamentous fungus protein.

Description

Technical Field

[0001] This invention relates to the field of Fusarium vesicatoria protein production technology. More specifically, this invention relates to a method for the resource recovery and utilization of waste liquid during the production of Fusarium vesicatoria protein. Background Technology

[0002] With the global shortage of protein resources and the increasing demand for sustainable protein sources, microbial proteins have received widespread attention as a novel alternative protein source. *Fusarium vesicatoria* (Venetian Fusarium) Fusariumvenenatum This fungus is a filamentous fungus rich in nutrients, high in protein, and with a well-balanced amino acid composition. As a filamentous fungus, its high protein yield and favorable amino acid composition have led to its widespread use in European and American markets (such as Quorn™). This fungus can efficiently accumulate cell protein through deep liquid aerobic fermentation, demonstrating promising industrialization potential.

[0003] The industrial preparation of mycelial protein typically involves steps such as fermentation, cell harvesting, nucleic acid removal and heat inactivation, and drying. However, these processes generate large amounts of organic wastewater, particularly at the following two key stages: 1. Fermentation waste liquid: refers to the fermentation supernatant produced when the mycelium is collected by plate and frame filtration after fermentation is completed.

[0004] 2. Waste liquid after nucleic acid removal treatment: refers to the liquid generated when mycelium is collected again after being heated and inactivated and degraded by nucleic acid.

[0005] These two types of waste liquid contain certain organic negative components and some nutrient residues. If discharged directly without treatment, they will not only cause environmental pressure but also increase production costs. Therefore, developing a method to recycle and reuse waste liquid is of great significance for promoting the green manufacturing of microbial proteins. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0007] To achieve these objectives and other advantages according to the present invention, a method for resource recovery and utilization of waste liquid from the production process of Fusarium vesicatoria protein is provided, wherein the waste liquid includes fermentation waste liquid and / or nucleic acid removal treatment waste liquid, the method comprising: The carbon source content in the waste liquid is detected. When the carbon source content in the waste liquid is lower than the preset concentration, the carbon source in the waste liquid is replenished to the preset concentration, and then Fusarium moniliforme TB6050 is inoculated. The above process is executed n times, where n≥1.

[0008] Preferably, when the waste liquid is fermentation waste liquid, n=1.

[0009] Preferably, when the waste liquid is nucleic acid removal treatment waste liquid, n=2.

[0010] Preferably, the preset concentration of the carbon source is 10~70g / L.

[0011] Preferably, the inoculation process of *Fusarium venetum* TB6050 includes: collecting spores prepared from fresh culture medium, counting the spores using a hemocytometer, and adding 1×10⁻⁶ spores per 100 mL of waste liquid. 6 Inoculate with 1 spore.

[0012] Preferably, the fermentation conditions are: 15~35℃, sterile air ventilation rate 0~1.5 vvm, pH 5~7, and culture at 50~350 rpm for 18~72h.

[0013] The present invention has at least the following beneficial effects: Fermentation of the waste liquid after nucleic acid removal treatment, supplemented with glucose at levels of 10–70 g / L, yielded a cell dry weight exceeding 19 g / L, with growth performance superior to that of standard culture media. This indicates that such waste liquid has good re-fermentation potential after appropriate carbon source regulation. Treatment with only carbon source supplementation (glucose) achieves high biomass without requiring nitrogen or other nutrients, demonstrating superior economic efficiency and ease of operation. This provides a low-cost, high-efficiency feasible path for the resource utilization of waste liquid. The fermentation waste broth showed some support during the initial fed-batch fermentation, but cell growth ceased after the second round of recycling, indicating a periodic limitation to its reusability. While reusable, it is not suitable for multiple rounds of continuous fermentation. Under the same fed-batch conditions, the waste broth treated with nucleic acid removal more effectively supported the growth of Fusarium tumefaciens strain TB6050, exhibiting significantly better fermentation performance and final biomass than the fermentation waste broth group, demonstrating higher resource utilization value.

[0014] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0015] Figure 1 The images are low-power microscopic images of typical samples after the first round of waste liquid recycling and fermentation. In the images, A shows the mycelial state after fermentation for 72 hours in freshly prepared control medium, and B shows the mycelial state after fermentation for 72 hours in a medium supplemented with carbon source from denucleated waste liquid. Figure 2 Images show the shake-flask state of typical samples after the second round of waste liquid recycling and fermentation. In the images, A represents the bacterial cell condition after the second round of fermentation waste liquid recycling, and B represents the bacterial cell condition after the second round of nucleic acid removal waste liquid recycling. Detailed Implementation

[0016] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0017] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified. Example

[0018] (1) Waste liquid collection and classification Fermentation waste liquid: The liquid obtained after separating the fermented bacteria by plate and frame filtration after the completion of the fermentation of Fusarium veitchii in a 5-ton tank; Nucleic acid removal waste liquid: The bacterial cells collected above are put into water and further treated with high temperature, nucleic acid degradation and inactivation, and then the filtrate is collected by plate and frame filtration again.

[0019] (2) Component analysis and resource utilization potential assessment The total carbon and total nitrogen content (e.g., NH4⁺, organic nitrogen) and metal ion composition (e.g., Mg²⁺) of the two types of waste liquids were determined separately. ＋ K ＋ Fe² ＋ Zn² ＋ (etc.), the results are shown in Tables 1 and 2 below; Table 1. Determination of total nitrogen, total phosphorus, and ammonia nitrogen content in the two types of waste liquids. Table 2. Determination of metal ion composition of the two types of waste liquids The above results indicate that both types of waste liquids retain a certain amount of available carbon sources, nitrogen sources and trace elements, and have the potential for re-fermentation; after nucleic acid removal treatment, there are no obvious antibacterial substances in the waste liquids, and the pH is suitable (about 6.5~7.0), which provides a basis for their resource utilization.

[0020] (3) Multi-round recycling experiment of carbon source supplementation for waste liquid Based on the recovered waste liquid, a treatment group and different control groups were set up to supplement carbon source (such as glucose) to a level of 40 g / L for fermentation evaluation. Fusarium tumefaciens TB6050 was used for shake-flask fermentation (500 mL flask volume, 110 mL culture medium per flask), and changes in cell biomass and fermentation broth composition were recorded. After fermentation, the residual glucose content in the culture medium was measured, and it was replenished again to 40 g / L, followed by high-temperature autoclaving. A second round of inoculation and fermentation was then initiated. This process was repeated for a third round to simulate the continuous recycling potential of the waste liquid. The first round of waste liquid recycling fermentation was recorded as n=1, and the value of n was increased by 1 for each subsequent recycling. A parallel control with standard fermentation medium was also included in each round of fermentation to compare and evaluate the impact of the waste liquid system on cell growth and metabolic capacity. The specific process is as follows: a) First-round recycling and utilization of waste liquid through fermentation The treatment groups included a fermentation waste liquid group, a fermentation waste liquid group with initial carbon source replenishment, a nucleic acid removal waste liquid group, and a nucleic acid removal waste liquid group with initial carbon source replenishment. In the fermentation waste liquid group and the nucleic acid removal waste liquid group with initial carbon source replenishment, the initial glucose concentration and total carbon and nitrogen content were measured. Then, carbon source replenishment was performed according to the nutrient levels of the standard fermentation medium (fermentation medium formula: (NH4)2SO4 1~10 g / L, MgSO4•7H2O 0.5~2 g / L, KCl 0.5~2 g / L, Na2SO4 0.5~2 g / L, KH2PO4 1~5 g / L, CaCO3 0.2~2 g / L, yeast extract 0.5~5 g / L, glucose 40 g / L). Carbon source replenishment was performed by supplementing with glucose to a mass concentration of 40 g / L to ensure the carbon source level was consistent with the standard fermentation medium. The control group was the standard fermentation medium group. The liquids from both the treatment and control groups were autoclaved and used as the first-round fermentation culture medium.

[0021] Both the treatment and control groups were inoculated with *Fusarium venetum* TB6050. The inoculation process included: collecting spores prepared from fresh culture medium, counting the spores using a hemocytometer, and adding 1×10⁻⁶ spores per 100 mL of waste liquid. 6 The spores were inoculated. The *Fusarium venetum* TB6050 has been disclosed in patent application number "2023108930309", entitled "Pyruvate decarboxylase gene FvPDC6 and its application in improving the mycelial protein yield of *Fusarium venetum*".

[0022] Both the treatment group and the control group were subjected to shake-flask fermentation after inoculation. The conditions for shake-flask fermentation were: 28℃, 180 rpm for 72 h.

[0023] After fermentation, the bacterial biomass and protein percentage content of the treatment group and the control group were measured respectively (the same measurement method was used in subsequent experiments).

[0024] Biomass and protein content determination: Fusarium tumefaciens TB6050 mycelia from fermentation culture were collected by vacuum filtration and washed three times with pure water. Subsequently, the mycelia were dried to constant weight in an electric thermostatic drying oven at 85℃ to determine the weight of the dried mycelia. This experiment was repeated three times, and the standard deviation was calculated. Protein content was determined using an elemental analyzer to measure the nitrogen content (%) of the dried mycelia, and the protein content was calculated according to the formula: Protein content (%) = Nitrogen content × 6.25.

[0025] Determination of glucose concentration and calculation of conversion rate: *Fusarium venetum* TB6050 fermented at 28℃ was centrifuged to remove mycelia, and the supernatant was collected and filtered through a 0.22 µm membrane to remove impurities. The cell-free supernatant was diluted 100-fold, and the glucose concentration was determined using a biochemical analyzer.

[0026] The results are as follows Figure 1 As shown in Table 3: Table 3. Data from the first round of fermentation (72 hours) for different culture media. Fermentation waste liquid Initial carbon source replenishment for fermentation waste liquid Nucleic acid removal waste liquid Initial carbon source replenishment for nucleic acid removal waste liquid Standard culture medium (control) Glucose content (g / L) in the culture medium after fermentation 0 0 0 0 13.5 Biomass (g / L) 10.56 13.25 2.29 19.73 8.65 Protein content (%) 48.4 56.7 52.4 25.41 37.23 The results show that both direct recycling of fermentation waste liquid and nucleic acid removal waste liquid, and carbon source supplementation of the above waste liquids, can effectively produce TB6050 cells. However, the accumulation capacity is significantly improved after carbon source supplementation. The waste liquid obtained from the nucleic acid removal process showed the best biomass accumulation capacity after carbon source supplementation, reaching 19.73 g / L.

[0027] b. Second-round recycling and fermentation of waste liquid In the first round of fermentation experiments, the waste liquid system with replenished carbon source significantly increased the biomass of the target strain TB6050, verifying the effectiveness of this treatment method in resource utilization. To further explore the applicability and stability of this method in the multi-round recycling process of waste liquid, the liquids from the initial carbon source replenishment group of fermentation waste liquid and the initial carbon source replenishment group of nucleic acid removal waste liquid collected after the first round of fermentation were selected as the basis for reuse.

[0028] The treatment groups included a fermentation waste liquid carbon source replenishment group and a nucleic acid removal waste liquid carbon source replenishment group. The fermentation waste liquid carbon source replenishment group involved removing bacterial residues from the initial carbon source replenishment group and then replenishing glucose to the target concentration of 40 g / L. The nucleic acid removal waste liquid carbon source replenishment group involved removing bacterial residues from the initial carbon source replenishment group (by centrifugation) and then replenishing glucose to the target concentration of 40 g / L. The control group remained the standard fermentation medium group. Both treatment and control groups underwent autoclaving and a second round of inoculation fermentation experiments.

[0029] The inoculum size and the shake-flask fermentation conditions for the treatment and control groups were the same as in the first round.

[0030] After fermentation, a biochemical analyzer was used to measure indicators such as cell biomass and residual sugar concentration in the treatment and control groups, respectively, to systematically evaluate the biomass accumulation capacity of the waste liquid system during continuous recycling. The results are as follows: Figure 2 As shown in Table 4: Table 4. Data from the second round of fermentation for 72 hours on different culture media. Secondary carbon source replenishment for fermentation waste liquid Secondary carbon source replenishment for nucleic acid removal waste liquid Standard culture medium (control) Glucose content (g / L) in the culture medium after fermentation 37 0 24 Biomass (g / L) / 5.96 6.52 The results above show that in the second round of fermentation, the fermentation waste liquid could no longer be cultured into cells, while the treatment of the denucleated waste liquid with carbon source replenishment still had a strong nutritional support capacity, and its biomass reached 5.96 g / L.

[0031] c. Third-round recycling of waste liquid through fermentation The results of the second round of experiments further demonstrated that the waste liquid after nucleic acid removal treatment still possessed a strong ability to support bacterial growth after carbon source replenishment, and strain TB6050 achieved a high biomass accumulation in this system. However, compared with the first round of fermentation, its cell dry weight had decreased significantly, suggesting that some key nutrients in the waste liquid may have been gradually depleted during the first two rounds of fermentation. To further explore the ultimate usability of this type of waste liquid, a third round of carbon source replenishment and fermentation experiments was conducted. The experiments were based on the carbon source replenishment group of the nucleic acid removal waste liquid collected after the second round of fermentation.

[0032] The treatment group included a group where the denucleated waste liquid was replenished with carbon source three times. In this group, the denucleated waste liquid was replenished with carbon source twice, and after removing bacterial residue, glucose was added again to the target mass concentration of 40 g / L. The control group was still the standard fermentation medium group. After autoclaving, the group was inoculated and fermented for the third time.

[0033] The inoculum size and the shake-flask fermentation conditions for the treatment and control groups were the same as in the first round.

[0034] After fermentation, the bacterial biomass and residual sugar concentration of the treatment and control groups were measured using a biochemical analyzer to evaluate the resource utilization potential and metabolic support capacity boundary of the denucleated waste liquid under multiple rounds of use. The results are shown in Table 5. Table 5. Data from the third round of fermentation for 72 hours on different culture media. Carbon source replenishment for nucleic acid removal waste liquid Standard culture medium (control) Glucose content (g / L) in the culture medium after fermentation 37 20 Biomass (g / L) / 7.13 The results above show that the nucleic acid-degraded waste liquid recycled in the third round could no longer effectively culture bacteria, indicating that it was severely deficient in nutrients and reached the limit of recycling under these conditions.

[0035] (4) Strategy of using nucleic acid removal waste liquid as an aqueous phase alternative water. The waste liquid after nucleic acid removal was used directly as the solvent (aqueous phase) of the fermentation medium. Various nutrients (including carbon source, nitrogen source, trace elements, etc.) were added according to the standard fermentation medium formula to prepare a complete fermentation medium as the medium for the first round of recycling. After the prepared medium was sterilized by high temperature and high pressure, the target strain TB6050 was inoculated and shake-flask fermentation was carried out to evaluate its ability to support cell growth and metabolism. After the fermentation was completed, based on the experience of the above treatment, the residual glucose content of the liquid obtained after cell separation was determined, and it was replenished to the level of 40 g / L to prepare the test medium for the next round of use. Then, the next round of fermentation evaluation was carried out (the inoculation amount and fermentation conditions of each round were the same as the first round in (3)). This strategy aims to verify the feasibility and recycling value of using the waste liquid after nucleic acid removal directly as fermentation water. The results are shown in Table 6: Table 6. Changes in cell dry weight during the first round of fermentation using the waste liquid after nucleic acid removal as the aqueous phase for the reused culture medium and subsequent fermentation rounds. First round of recycling Second round of recycling Third round of recycling Standard culture medium (control) Glucose content (g / L) in the culture medium after fermentation 11.7 35 33 21 Biomass (g / L) 6.55 2.53 0.19 6.25 The results above show that, compared with the fermentation system obtained by supplementing only carbon source in the waste liquid after nucleic acid removal treatment (as mentioned above), the total dry weight of the cells in the treatment group supplemented with all nutrients (including carbon source, nitrogen source and trace elements) is less than that in the treatment group supplemented with only carbon source.

[0036] (5) The carbon source for the waste liquid after nucleic acid removal is replenished and recycled in a single fermentation in a 5L fermenter. Based on the waste liquid after nucleic acid removal treatment, a treatment group with supplemented carbon source (such as glucose) to 60 g / L and a control group with standard fermentation medium were set up for fermentation evaluation. Fusarium tumefaciens TB6050 was used for inoculation and fermentation was carried out in a 5L fermenter. Changes in cell biomass and fermentation broth composition were recorded.

[0037] The standard fermentation medium formula includes: (NH4)2SO4 5~15g / L, MgSO4•7H2O 0.5~2g / L, KCl 0.5~2g / L, Na2SO4 0.5~2g / L, KH2PO4 1~5g / L, CaCO3 0.2~2g / L, yeast extract 0.5~5g / L, and glucose 60g / L.

[0038] The inoculation method for Fusarium venetum TB6050 was the same as that used in the shake-flask fermentation described above.

[0039] Fermentation conditions were: 28℃, sterile air flow rate of 1.0 vvm, pH 6.0, and culture at 350 rpm for 48 h.

[0040] Biomass, glucose content, and protein content in the fermenter were measured at 24, 36, and 48 hours, and sugar consumption and carbon conversion rate were calculated simultaneously. Sugar consumption was calculated using the following formula: Sugar consumption = Initial glucose concentration - Glucose concentration at measurement. Carbon conversion rate (g / g) was calculated using the following formula: Carbon conversion rate = Biomass / (Initial glucose concentration - Glucose concentration at measurement). The results are shown in Table 7. Table 7 Data on the recovery and utilization of different culture media after 48 hours of fermentation sample Biomass g / L Sugar consumption g / L Carbon conversion rate (g / g) Protein content % F24 4.82 18 0.27 53.35 T24 5.25 16 0.33 55.75 F36 9.06 34 0.27 58.55 T36 12.77 31 0.41 58.94 F48 11.58 45 0.26 55.81 T48 16.62 46 0.36 55.27 Note: F24, F36, and F48 represent the test data of the standard fermentation medium at 24, 36, and 48 hours, respectively; T24, T36, and T48 represent the detection data of culture medium recycled from waste liquid after nucleic acid removal treatment at 24, 36, and 48 hours, respectively.

[0041] Based on the comparative data from 48 hours of fermentation in a 5L fermenter (Table 7), the recycled culture medium prepared using the carbon source supplemented from the waste liquid after nucleic acid removal treatment (Group T) showed superior performance compared to the standard fermentation medium (Group F) in terms of both cell growth and raw material utilization efficiency. Specifically, at the 24, 36, and 48-hour fermentation times, the biomass accumulation of Group T (5.25, 12.77, and 16.62 g / L, respectively) was significantly higher than that of Group F (4.82, 9.06, and 11.58 g / L, respectively), demonstrating stronger support for cell growth. Simultaneously, the carbon conversion rates of Group T at each stage (0.33, 0.41, and 0.36 g / g) were also significantly higher than those of Group F (0.27, 0.27, and 0.26 g / g), indicating that the glucose conversion to cell volume was more efficient and economical in the recycling system. The final sugar consumption levels of the two groups were similar (Group F 45 g / L, Group T 46 g / L), and there was no significant difference in protein content (approximately 55%-59%), indicating that the recycling of the waste liquid did not affect the overall metabolic intensity and cell protein quality. In conclusion, after carbon source supplementation, the nucleic acid removal waste liquid can not only serve as a highly efficient fermentation substrate, but also surpass standard culture media in terms of yield and conversion rate, demonstrating its excellent potential for resource recovery and economic viability.

[0042] (6) Construction of waste liquid reuse model and analysis of theoretical emission reduction effect Taking a 5-ton fermentation system as an example, without wastewater reuse, each production cycle generates 3 tons of fermentation wastewater and 1.7 tons of nucleic acid removal wastewater, for a total of 4.7 tons of the two types of wastewater per cycle. The theoretical amount of the two types of wastewater generated over four consecutive production cycles is: (3 + 1.7) × 4 = 18.8 tons.

[0043] Under the recycling strategy described in this invention: fermentation waste liquid is set to be reused a maximum of 1 time; nucleic acid removal waste liquid is set to be reused a maximum of 2 times. Material balance results for four production cycles according to the above reuse rules (Table 8) show that the final actual discharge of the two types of wastewater is 7.7 t. Compared with conventional processes, theoretical calculations based on the reuse frequency setting show that the discharge of these two types of wastewater can be reduced by approximately 59.04%.

[0044] Table 8 shows the generation of two types of waste liquid in four production cycles under the waste liquid recycling model (taking a 5-ton fermenter as an example, unit: t). Production cycle Fermentation waste liquid generation Fermentation waste liquid treatment methods Nucleic acid removal waste liquid generation Nucleic acid removal waste liquid treatment methods Actual emissions during the period Period 1 3 For the next cycle 1.7 Delayed reuse in subsequent cycles 0 Period 2 3 Reuse limit reached, emissions 1.7 Recycled in subsequent cycles 3 Period 3 3 For the next cycle 1.7 For the next cycle 0 Period 4 3 Emissions Treatment 1.7 Emissions Treatment 4.7 Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for the resource recovery and utilization of waste liquid in the production process of Fusarium vesicatoria protein, characterized in that, The waste liquid includes fermentation waste liquid and / or nucleic acid removal treatment waste liquid, and the method includes: The carbon source content in the waste liquid is detected. When the carbon source content in the waste liquid is lower than the preset concentration, the carbon source in the waste liquid is replenished to the preset concentration, and then Fusarium moniliforme TB6050 is inoculated for fermentation. The above process is executed n times, where n≥1.

2. The method for resource recovery and utilization of waste liquid in the production process of Fusarium vesicatoria protein as described in claim 1, characterized in that, When the waste liquid is fermentation waste liquid, n=1.

3. The method for resource recovery and utilization of waste liquid in the production process of Fusarium vesicatoria protein as described in claim 1, characterized in that, When the waste liquid is nucleic acid removal treatment waste liquid, n=2.

4. The method for resource recovery and utilization of waste liquid in the production process of Fusarium vesicatoria protein as described in claim 1, characterized in that, The preset concentration of the carbon source is 10~70g / L.

5. The method for resource recovery and utilization of waste liquid in the production process of Fusarium vesicatoria protein as described in claim 1, characterized in that, The inoculation process for Fusarium venetum TB6050 includes: collecting spores prepared from fresh culture medium, counting the spores using a hemocytometer, and adding 1×10⁻⁶ spores per 100 mL of waste liquid. 6 Inoculate with 1 spore.

6. The method for resource recovery and utilization of waste liquid in the production process of Fusarium vesicatoria protein as described in claim 1, characterized in that, The fermentation conditions are: 15~35℃, sterile air ventilation rate 0~1.5 vvm, pH 5~7, and culture at 50~350 rpm for 18~72 h.

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