Method for preparing biogas slurry nanofiltration permeate culture medium and using biogas slurry nanofiltration permeate culture medium in continuous production of single-cell protein

Abstract

The invention relates to the technical field of waste resource utilization, in particular to preparation of a biogas slurry nanofiltration permeate culture medium and a method for applying the biogas slurry nanofiltration permeate culture medium to continuous production of single-cell protein. The method comprises the following steps: carrying out matrix filtration on biogas slurry obtained by carrying out anaerobic fermentation on livestock and poultry manure, and carrying out membrane filtration on filtrate of the biogas slurry after the matrix filtration; the matrix filtration is enhanced by adopting a coagulant, the membrane filtration is carried out step by step by adopting ultrafiltration and nanofiltration, and the obtained permeate is subjected to C / N and pH regulation. The biogas slurry nanofiltration permeate culture medium provided by the invention is suitable for growth and metabolism of candida utilis, the protein synthesis efficiency is remarkably improved, the nanofiltration permeate is high in purity, interference of infectious microbes and impurities is reduced, the nutritional value and safety of a product are improved, and efficient production of single-cell protein and resource utilization of biogas slurry can be realized.

Description

Technical Field

[0001] The present invention relates to the technical fields of waste resource utilization and feed production, and in particular to a method for preparing a biogas slurry nanofiltration permeate culture medium and using the same for continuous production of single-cell protein. Background Art

[0002] As an important part of food security, animal husbandry has an impact on the environment due to its over-reliance on land resources and high carbon emissions. Finding new feed production methods is of great significance to improving food security, reducing carbon emissions and alleviating land dependence.

[0003] Single-cell protein (SCP) is a high-quality protein feed produced by microbial fermentation. It boasts advantages such as rapid synthesis, a low land and sunlight requirement, and efficient wastewater recycling. SCP production can effectively alleviate environmental pressures and recover nutrients from otherwise difficult-to-use wastewater, playing a crucial role in achieving sustainable development in the livestock industry. However, insufficient SCP production limits its feasibility for industrial application, primarily due to the immaturity of production technology and the lack of efficient and stable production methods. At present, there are patent applications for single-cell protein production, such as CN115594310A involving a method for producing single-cell protein for feed from livestock and poultry manure and biogas slurry, and CN113913309A involving an alkali-resistant yeast and its use in producing single-cell protein from biogas slurry. They disclose that biogas slurry can be used for protein synthesis; however, this type of technology currently has major defects, mainly: 1) production using wastewater or biogas slurry raw liquid, resulting in high impurities, low protein content, poor palatability, and a high number of pathogens; 2) lack of process conditions, and specific parameters such as culture, stirring, C / N ratio, pH, etc. are unclear, resulting in a lack of industrial production capacity for single-cell protein; 3) current batch processing cannot achieve continuous production, and production efficiency is low. Summary of the Invention

[0004] To address the above technical problems, the present invention provides a method for preparing a biogas slurry nanofiltration permeate culture medium and its use in the continuous production of single-cell protein. The biogas slurry nanofiltration permeate culture medium provided by the present invention is suitable for the growth and metabolism of Candida utilis, significantly improving protein synthesis efficiency. Furthermore, the nanofiltration permeate has high purity, reducing interference from foreign bacteria, improving the nutritional value of the product, and enabling efficient production and resource utilization of single-cell protein.

[0005] According to the present invention, the technology for using single-cell protein to recover nutrients from wastewater and produce high-quality feed protein is currently in its infancy. However, several key challenges remain: 1) Biogas slurry (or other wastewater) contains a large amount of impurities and bacteria. Using it as a raw material for protein production results in poor product palatability, a high concentration of bacteria (especially pathogenic microorganisms), and interference with the normal protein synthesis process of protein-producing bacteria, resulting in reduced yields. This requires costly high-temperature sterilization to prevent antagonistic and direct toxic effects from these bacteria. 2) Production process parameters are imperfect, and without a clear definition of sterilization or maintaining a sterile environment after sterilization, protein-producing bacteria are cultivated solely through inoculation, resulting in low protein synthesis efficiency. 3) Current batch production models result in unstable yields. 4) The performance of protein-producing bacteria is low. 5) There is no suitable resource-resource utilization method for biogas slurry nanofiltration permeate; currently, it is only treated to meet discharge standards, resulting in wasteful resources. However, addressing these multiple technical challenges and further technical research and optimization still face numerous challenges and obstacles.

[0006] In a first aspect, the present invention provides a method for preparing a biogas slurry nanofiltration permeate culture medium, comprising: subjecting biogas slurry obtained by anaerobic fermentation of livestock and poultry manure to matrix filtration, and subjecting the biogas slurry filtrate to membrane filtration after the matrix filtration; the matrix filtration adopts a coagulant to enhance the matrix filtration, the membrane filtration adopts ultrafiltration and nanofiltration to perform step-by-step filtration, and the C / N and pH of the obtained permeate are adjusted.

[0007] This method subjects anaerobic fermentation of livestock and poultry manure to a coagulant-enhanced matrix filtration process. Direct mixing of the coagulant with the matrix removes impurities from the biogas slurry, effectively improving the efficiency of subsequent membrane separation processes and reducing membrane fouling. Ultrafiltration and nanofiltration are then performed to optimize biogas slurry treatment conditions, resulting in a permeate richer in available nutrients and contributing to increased single-cell protein yield and quality. After the coagulant-enhanced matrix filtration fails, the biogas slurry can be directly composted to avoid secondary contamination.

[0008] Preferably, in the matrix material, the coagulant is chitosan or modified starch, and the amount of the coagulant added is 1-5 kg / m 3 (based on the volume of the substrate), preferably 3.5 kg / m 3 ; The matrix material also includes one or more of quartz sand, river sand, and crop straw.

[0009] Preferably, the ultrafiltration uses a PVDF hollow fiber UF membrane with an operating pressure of 0.1-0.3 MPa and a flow rate of 38-42 L / h.

[0010] Preferably, the nanofiltration uses an ESNA1-4040 spiral NF membrane with an operating pressure of 0.3-0.6 MPa and a flow rate of 28-32 L / h to obtain a permeate.

[0011] The present method for preparing biogas slurry nanofiltration permeate, through matrix filtration, ultrafiltration, and nanofiltration under specific conditions and parameters, produces a permeate rich in small-molecule organic matter and trace elements, optimizing the bacterial growth environment and increasing protein yield and quality. Simultaneously, by optimizing the C / N ratio and pH, the permeate is more suitable for the growth and metabolism of Candida utilis, significantly improving protein synthesis efficiency. Compared to the original biogas slurry, the nanofiltration permeate has a higher purity, reduces interference from foreign bacteria, and improves the nutritional value of the product. This method enables the efficient production and resource utilization of single-cell protein.

[0012] More preferably, the C / N ratio of the permeate is adjusted to 2-4 using molasses or glucose, and the pH is adjusted to 5-7 using 2-3 mol / L hydrochloric acid.

[0013] In this invention, by adjusting the C / N ratio and pH of the permeate, the production environment for single-cell protein can be optimized, providing the nutritional structure required by Candida utilis, thereby promoting its growth and protein synthesis. Under optimal conditions, the metabolic activity of the microorganism can be more effectively promoted, increasing protein yield and growth rate, while also avoiding the adverse effects of excessively high or low C / N ratios and pH on microbial growth, thereby improving production efficiency.

[0014] Preferably, molasses or glucose is added to adjust the C / N ratio of the permeate to 2.5-3.5, and 2-3 mol / L hydrochloric acid is used to adjust the pH of the permeate to 5-7. The addition of glucose helps provide an additional carbon source and nutrients, further increasing protein yield. Meanwhile, adjusting the pH of the culture environment with hydrochloric acid helps maintain optimal yeast growth.

[0015] The present invention provides a method for preparing a culture medium for optimizing biogas slurry nanofiltration permeate. The resulting biogas slurry nanofiltration permeate can provide ideal nutrition and growth conditions for single-cell protein production and eliminate impurities and a large number of foreign bacteria. The present invention subjects biogas slurry to matrix filtration, hollow fiber UF membrane ultrafiltration, and spiral NF membrane nanofiltration. The resulting permeate, after adjusting its C / N ratio and pH value, can provide ideal nutritional conditions for single-cell protein production, optimize the growth environment of Candida utilis, and promote its metabolic activity and protein synthesis. Through process optimization, protein synthesis efficiency can be effectively improved, interference from foreign bacteria can be reduced, and sterilization operations that increase costs can be avoided, ultimately achieving efficient, stable, and continuous single-cell protein production.

[0016] In a second aspect, the present invention provides a biogas slurry nanofiltration permeate, which is prepared by the above-mentioned biogas slurry nanofiltration permeate preparation method.

[0017] The present invention uses biogas slurry nanofiltration permeate as the mother liquor for single-cell protein production. This reduces impurities in the biogas slurry and enriches it with small organic molecules, which is beneficial for the growth of single-cell protein. Furthermore, the number of harmful bacteria is low even without sterilization. This invention enables a continuous production model, achieving stable protein yields and addressing the issue of unstable yields in batch production models.

[0018] In a third aspect, the present invention provides the use of the biogas slurry nanofiltration permeate obtained by the above preparation method in the production of single cell protein.

[0019] The present invention utilizes membrane concentration technology to concentrate biogas slurry, and uses the clean permeate rich in small molecule nutrients for continuous single-cell protein production. It has the characteristics of stable production performance, high protein content, and few foreign bacteria. The nanofiltration permeate used is rich in small molecule organic matter, medium and trace elements, and phosphates, which is more in line with the basic growth and reproduction conditions of bacteria, and thus has better bacterial growth conditions and protein production characteristics. In addition, the membrane-concentrated permeate has a high degree of purity, which reduces the interference of foreign bacteria, improves the palatability and nutritional value of the product, and the present invention improves the protein content and protein yield of the product. By utilizing the biogas nanofiltration permeate as a resource and converting it into high-value single-cell protein, not only is the waste of resources avoided, but the high-value utilization of biogas slurry is also achieved, opening up a new path for biogas slurry treatment and resource utilization.

[0020] In a fourth aspect, the present invention provides a method for producing single-cell protein, wherein the biogas slurry nanofiltration permeate culture medium obtained by the above-mentioned preparation method is used to continuously ferment and cultivate single-cell protein.

[0021] Preferably, a bacterial suspension prepared with Candida utilis is inoculated into the biogas slurry nanofiltration permeate culture medium, and the volume ratio of the bacterial suspension to the biogas slurry nanofiltration permeate is 8-10:100; the charging coefficient (η=Vactual / Vtotal) is 8 / 25-15 / 25, preferably 10 / 25.

[0022] More preferably, the incubation temperature is 25-40°C, preferably 30°C.

[0023] More preferably, the fermentation liquid residence time is 36-72 h, preferably 36-48 h, and the inlet and outlet liquid flow rates per unit volume are 13.9-27.8 L / h, preferably 14-21 L / h.

[0024] More preferably, the stirring rate of the continuous fermentation is 100-200 rpm / mi, preferably 150 rpm / mi.

[0025] In this invention, adjustments to the initial inoculum size, loading factor, temperature, and agitation rate effectively increase single-cell protein yield, boost cell concentration, enhance stability, and reduce operational failure rates (i.e., microbial inhibition and reduced activity). By optimizing a series of parameters for continuous production, single-cell protein production can be achieved without sterilizing the culture medium. (On the one hand, the culture medium preparation method of this invention eliminates most contaminants; on the other hand, the remaining bacteria promote, rather than inhibit, single-cell protein production by Candida utilis.) Compared to batch production, this significantly reduces operating costs, eases operational complexity, and enhances the stability of the fermentation system. Finally, this set of parameters produces a protein with a suitable nutritional profile and good palatability, fully complying with the requirements of the "Feed Hygiene Standard" (GB 13078-2017).

[0026] The nanofiltration permeate provided by the present invention is rich in small-molecule organic matter, trace elements, and phosphates, which better meet the basic growth and reproduction conditions of bacteria, thereby having better bacterial growth conditions and protein production characteristics. In addition, the membrane-concentrated permeate has high purity, reducing the interference of miscellaneous bacteria, improving the palatability and nutritional value of the product. Compared with the permeate of raw biogas slurry and other membrane separation processes, the protein content of the nanofiltration permeate is increased by 30% and the protein yield is increased by 45% ( Figure 1 The process conditions of culture temperature of 30°C, bacterial suspension inoculation of 10% (v / v), C / N ratio of 3, and pH of 6 are more conducive to the growth metabolism and protein synthesis of Candida utilis. At the same time, it avoids the problems of high energy consumption and large nutrient loss caused by high temperature sterilization. The protein content of the product increased by 40% and the protein yield increased by 50% ( Figure 2 Through a continuous production model, the present invention achieves stable protein yields, solving the problem of low and unstable yields in batch production models. The present invention utilizes the biogas slurry nanofiltration permeate as a resource, converting it into high-value single-cell protein. This not only avoids resource waste but also achieves high-value utilization of biogas slurry, opening up new avenues for biogas slurry treatment and resource utilization. Using Candida utilis and a non-sterilization scheme, compared to a sterilization scheme and a method using indigenous bacteria to synthesize protein, the relative abundance of major pathogens was reduced by over 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1This figure shows the protein yield and content of Candida utilis cultured under different sterilization conditions for the biogas slurry provided in Example 1 and Comparative Example 1, namely, membrane filtration products obtained under non-sterilized (A) and sterilized (B) conditions. UF, NF, and RO represent ultrafiltration, nanofiltration, and reverse osmosis, respectively. "Cent" represents the concentrate, and "Permeate" represents the permeate.

[0029] Figure 2 The biomass and protein yield of Candida utilis under different initial C / N ratios (A) and pH (B) conditions provided in Example 2 of the present invention and Comparative Examples 2-9.

[0030] Figure 3 A schematic diagram of a process provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0031] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0032] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to form one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed in the present invention.

[0033] Where specific techniques or conditions are not specified in the examples of the present invention, the techniques or conditions described in the literature in the field or in the product specifications were followed. All devices, instruments, reagents, etc. used, where the manufacturer is not specified, are conventional products available through regular channels. All experimental reagents and raw materials involved are commercially available, and all reagents are analytically pure.

[0034] In the following examples, the main experimental equipment involved are: centrifuge, continuous microbial fermentation tank (automatic temperature control required), ultrafiltration and nanofiltration membrane devices, and peristaltic feed pump.

[0035] In the examples of the present invention, the Candida utilis used was purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0036] Example 1 This embodiment provides a biogas slurry nanofiltration permeate and a method for continuously producing single-cell protein using the biogas slurry nanofiltration permeate. Figure 3 As shown, the following steps are taken: a) Select the biogas slurry from wet anaerobic fermentation of chicken manure and add chitosan at a rate of 3.5 kg / m 3 The biogas slurry was thoroughly mixed with corn straw in a certain proportion and stirred to form a filtration matrix. After filtering through the matrix, it was filtered using a PVDF hollow fiber UF membrane at an operating pressure of 0.2 MPa and a flow rate of 40 L / h to obtain an ultrafiltration concentrate and permeate. The ultrafiltration permeate was filtered using an ESNA1-4040 spiral wound NF membrane at an operating pressure of 0.5 MPa and a flow rate of 30 L / h to obtain a nanofiltration concentrate and permeate.

[0037] b) Adjusting the properties of the nanofiltration permeate: Glucose (powder) was added to adjust the C / N ratio to 3, and the pH of the nanofiltration permeate was adjusted to 6 using 2 mol / L hydrochloric acid.

[0038] c) Protein-producing bacterial culture: Inoculate culture medium (10% tryptone, 10% glucose, 5% yeast extract powder, solid medium supplemented with 2% agar) with Candida utilis and expand to the logarithmic phase at 30°C. Centrifuge the culture at 180 rpm for 5 minutes. Add the precipitate to sterile water to prepare a bacterial suspension with an OD value of 1.5 ± 0.3.

[0039] d) inoculating the nanofiltration permeate with the bacterial suspension at 10% (v / v) to form a fermentation broth, and incubating the fermentation broth in a closed continuous reactor for protein synthesis with a residence time of 72 hours and a loading volume of 150 / 250 of the reactor volume. The culture temperature was 30°C and agitation was performed at 150 rpm during the culture.

[0040] e) The fermentation effluent from the reactor is centrifuged in a centrifuge at 180 rpm, and the solid precipitate is dried in an oven at 60° C. until the moisture content is below 7%, thereby obtaining a dried single-cell protein product.

[0041] The single cell protein product was tested for biological yield, protein yield, and protein content. The test method was as follows. The test results of the embodiment are as follows: Figure 1 (A) shows "unsterilized" treatments, including raw biogas slurry and various concentrates / permeates used as fermentation solutions. Results show that the biomass yield, protein yield, and protein content of the unsterilized treatment were all higher than those of the sterilized treatment. The biogas slurry and UF concentrate had the highest biomass yield and protein yield, followed by the NF permeate. The NF permeate also had the highest protein content. However, testing revealed that the low protein content of the single-cell protein products from the raw biogas slurry and UF concentrate was primarily due to high impurity levels, which reduced product quality and palatability. Overall, the NF permeate performed best.

[0042] Comparative Example 1 This comparative example provides a method for continuously producing single-cell protein. The method is the same as that in Example 1, except that the non-sterilized treatment method is replaced by sterilization, thereby removing the interference of the original bacteria in the biogas slurry. The comparative example test results are shown in FIG. Figure 1 (B) shows "sterilization". The results show that the "sterilization" operation resulted in significantly lower biological yield, protein yield, and protein content than in Example 1. The "sterilization" operation is to sterilize the yeast solution at 121° C. and 103.4 KPa for 30 minutes.

[0043] Example 2 This embodiment provides a biogas slurry nanofiltration permeate and a method for continuously producing single-cell protein using the biogas slurry nanofiltration permeate. The method is the same as that of Example 1, except that the fermented yeast liquid is limited to the NF permeate. The test results of Example 2 are as follows: C / N=3 and Figure 2 (B) pH = 6.

[0044] Comparative Examples 2-5 This comparative example provides a biogas slurry nanofiltration permeate and a method for continuously producing single cell protein using the biogas slurry nanofiltration permeate. The method is the same as that of Example 2, except that the C / N ratio in step b is changed to 1, 5, 7 and 9 respectively. The test results are shown in FIG. Figure 2 (A) shown.

[0045] The method for changing the C / N ratio is to add glucose to adjust the yeast solution.

[0046] The results showed that the C / N ratio setting conditions of Comparative Examples 2-5 resulted in lower biomass yield, protein yield, and protein content than those of the Examples.

[0047] Comparative Examples 6-9 This comparative example provides a biogas slurry nanofiltration permeate and a method for continuously producing single cell protein using the biogas slurry nanofiltration permeate, using the same method as Example 2, except that the pH in step b is changed to 5, 7, 8 and 9 respectively. The test results are as follows Figure 2 (B)

[0048] The method for changing the C / N ratio is to add 2 mol / L hydrochloric acid to adjust the yeast solution.

[0049] The results showed that the pH setting conditions of Comparative Examples 6-9 resulted in lower biomass yield, protein yield, and protein content than those of the Examples.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing a biogas slurry nanofiltration permeate culture medium, characterized in that: include: The biogas slurry obtained by anaerobic fermentation of livestock and poultry manure is subjected to matrix filtration, and the biogas slurry filtrate is subjected to membrane filtration after the matrix filtration; The matrix filtration adopts coagulant to strengthen the matrix filtration, the membrane filtration adopts ultrafiltration and nanofiltration to perform step-by-step filtration, and the obtained permeate is subjected to C / N and pH adjustment.

2. The method for preparing the biogas slurry nanofiltration permeate culture medium according to claim 1, wherein In the matrix material, the coagulant is chitosan or modified starch, and the addition amount of the coagulant is 1~5 kg / m 3 , preferably 3.5 kg / m 3 ; The matrix material also includes one or more of quartz sand, river sand, and crop straw; The ultrafiltration uses a PVDF hollow fiber UF membrane with an operating pressure of 0.1-0.3 MPa and a flow rate of 38-42 L / h; The nanofiltration uses an ESNA1-4040 spiral NF membrane with an operating pressure of 0.3-0.6 MPa and a flow rate of 28-32 L / h to obtain a permeate.

3. The method for preparing a biogas slurry nanofiltration permeate culture medium according to claim 1 or 2, wherein: The C / N ratio of the permeate is adjusted to 2-4 using molasses or glucose, and the pH is adjusted to 5-7 using 2-3 mol / L hydrochloric acid.

4. A biogas slurry nanofiltration permeate culture medium, characterized in that: The biogas slurry nanofiltration permeate culture medium is prepared by the method for preparing the biogas slurry nanofiltration permeate culture medium according to any one of claims 1 to 3.

5. Use of the biogas slurry nanofiltration permeate culture medium obtained by the preparation method according to any one of claims 1 to 3 in the production of single-cell protein.

6. A method for producing single-cell protein, characterized in that: The biogas slurry nanofiltration permeate culture medium obtained by the preparation method according to any one of claims 1 to 3 is subjected to continuous fermentation to cultivate single-cell protein.

7. The method for producing single-cell protein according to claim 6, wherein: A bacterial suspension prepared with Candida utilis is inoculated into the biogas slurry nanofiltration permeate culture medium, with a volume ratio of the bacterial suspension to the biogas slurry nanofiltration permeate being 8-10:100; and a charging factor being 8 / 25-15 / 25, preferably 10 / 25.

8. The method for producing single-cell protein according to claim 6 or 7, characterized in that: The incubation temperature is 25-40°C, preferably 30°C.

9. The method for producing single-cell protein according to any one of claims 6 to 8, characterized in that: The fermentation liquid residence time is 36-72 hours, preferably 36-48 hours, and the inlet and outlet liquid flow rates per unit volume are 13.9-27.8 L / h, preferably 14-21 L / h.

10. The method for producing single-cell protein according to any one of claims 6 to 9, characterized in that: The stirring rate of continuous fermentation is 100-200 rpm / mi, preferably 150 rpm / mi.

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