A strain of high ammonia nitrogen tolerant yeast and its application in biogas slurry resource treatment

Abstract

The present application belongs to the field of environmental biotechnology and resource recycling technology, and provides a high-ammonia-nitrogen-tolerant yeast strain and its application in biogas slurry resource treatment. The high-ammonia-nitrogen-tolerant yeast strain is named Torulaspora delbrueckii L12345, which was preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 13, 2026, and the address is No. 1, Yitian West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No. 39123. The present application realizes efficient treatment of biogas slurry and simultaneous production of single-cell protein, reduces the treatment cost, improves the economic benefit, and provides a new technology for clean and high-value utilization of biogas slurry.

Description

Technical Field

[0001] This invention belongs to the field of environmental biotechnology and resource recycling technology, specifically relating to a strain of high ammonia nitrogen-tolerant yeast and its application in the resource-based treatment of biogas slurry. Background Technology

[0002] Currently, biogas slurry treatment methods mainly include physical separation, chemical sedimentation, agricultural application, and biological treatment. While agricultural application can recover nutrients, heavy metals, pathogens, and excessively high salinity in biogas slurry may pose long-term threats to soil and groundwater, and seasonal disposal challenges exist. Physicochemical methods are costly and struggle to achieve effective resource recovery. Biological treatment, particularly utilizing microorganisms to transform nutrients in biogas slurry into high-value products, is considered a promising clean and resource-efficient treatment approach.

[0003] Single-cell protein (SCP) refers to protein resources extracted from microorganisms such as bacteria, yeast, fungi, or microalgae. It is characterized by high protein content, rapid growth, and independence from climate and arable land limitations, making it a potential supplement to feed protein and even future food protein. Utilizing nutrient-rich biogas slurry to cultivate microorganisms for SCP production can achieve synergistic pollution control and resource recovery. However, biogas slurry generally exhibits high ammonia nitrogen concentrations (often exceeding 2000 mg / L) and low carbon-to-nitrogen ratios (C / N). High concentrations of ammonia nitrogen (especially free ammonia) strongly inhibit and toxicize most microorganisms; while the low C / N ratio limits the assimilation and utilization of nitrogen by microorganisms, resulting in slow microbial growth and low biomass yield. This has become a technical bottleneck restricting the efficient production of SCP using biogas slurry as a substrate.

[0004] While existing technologies utilize yeast to treat organic wastewater, there are few reports on specific yeast strains and their corresponding processes that can tolerate extremely high ammonia nitrogen concentrations (e.g., 4000 mg / L) and efficiently convert biogas slurry into SCP (concentrated organic solvent) under optimized conditions. Common solutions either involve diluting the biogas slurry, increasing treatment costs and volume, or searching for tolerant strains but failing to address their growth limitations in high-ammonia-nitrogen, low-C / N biogas slurry. Therefore, developing a strain and process capable of directly and efficiently treating high-ammonia-nitrogen raw biogas slurry and converting it into high-value SCP is of great significance for promoting the resource utilization of biogas slurry. Summary of the Invention

[0005] To address the challenge of efficiently utilizing high-ammonia-nitrogen biogas slurry for single-cell protein production by microorganisms in existing technologies, this invention aims to provide a high-ammonia-nitrogen-tolerant yeast strain and its application in biogas slurry resource utilization. This enables efficient biogas slurry treatment and simultaneous single-cell protein production, reducing treatment costs, increasing economic benefits, and providing a new technology for the clean and high-value utilization of biogas slurry.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, this invention provides a high-ammonia-nitrogen-tolerant yeast strain named *Torulaspora delbrueckii* L12345, which was deposited on January 13, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39123. This strain exhibits tolerance to ammonia nitrogen levels up to 4000 mg / L and can grow in a culture medium primarily composed of high-ammonia-nitrogen biogas slurry, converting the ammonia nitrogen and organic matter therein to synthesize cell proteins.

[0008] Secondly, the present invention provides the application of the above-mentioned high ammonia nitrogen-tolerant yeast in the resource utilization treatment of biogas slurry.

[0009] Preferably, the application includes inoculating Delburo's spore-forming yeast L12345 into a culture medium with high ammonia nitrogen biogas slurry as the main substrate for fermentation culture to obtain single-cell protein products.

[0010] More preferably, the ammonia nitrogen concentration of the high ammonia nitrogen biogas slurry is 3000 mg / L.

[0011] More preferably, the culture medium is a biogas slurry-molasses mixed culture medium with added molasses, wherein the carbon-nitrogen ratio (C / N) of the biogas slurry-molasses mixed culture medium is 3.0~15.0 and the initial pH value is 4.0~9.0.

[0012] More preferably, the fermentation culture is carried out aerobically for 1 to 5 days at a temperature of 30 ℃ and a shaking speed of 180 rpm.

[0013] Thirdly, the present invention provides a method for producing single-cell protein by treating biogas slurry with the above-mentioned high ammonia nitrogen-tolerant yeast, comprising the following steps:

[0014] S1. Pretreatment of biogas slurry: Collect biogas slurry and remove large suspended solids to obtain pretreated biogas slurry;

[0015] S2. Preparation of biogas slurry-molasses mixed culture medium: Add molasses to the pretreated biogas slurry, adjust the carbon-nitrogen ratio (C / N) of the biogas slurry-molasses mixed culture medium to 3.0~15.0 (preferably 9.0), adjust the initial pH of the mixed culture medium to 4.0~9.0 (preferably 6.0), and then sterilize it;

[0016] S3. Inoculation and culture: The seed culture of *Cytomyces delbrueckii* L12345 in the logarithmic growth phase is inoculated into sterilized biogas slurry-molasses mixed medium at a volume inoculation ratio of 5% to 30% (preferably 20%). The culture is carried out aerobically at a temperature of 30 ℃ and a shaking speed of 180 rpm for 1 to 5 days (preferably 4 days).

[0017] S4. Product Harvesting: After the culture is completed, the fermentation broth is separated into solid and liquid components, and the bacterial biomass is collected. After washing and drying, the single-cell protein product is obtained.

[0018] Preferably, the ammonia nitrogen concentration of the biogas slurry in step S1 is 3000 mg / L.

[0019] Preferably, the molasses in step S2 is sugarcane molasses.

[0020] Fourthly, the present invention provides the use of single-cell protein products produced according to the above method in animal feed or food additives.

[0021] Compared with the prior art, the present invention has the following significant advantages:

[0022] 1. The strain exhibits outstanding tolerance and strong applicability: The selected strain, *Cytomyces delbrueckii* L12345, can tolerate ammonia nitrogen up to 4000 mg / L. It can directly treat raw biogas slurry with high ammonia nitrogen concentration or biogas slurry that has only undergone simple pretreatment, without the need for large-scale dilution, thus reducing the treatment volume and subsequent costs.

[0023] 2. The process is highly innovative and solves growth limitations: It creatively proposes to adjust the C / N ratio of biogas slurry by adding inexpensive and readily available molasses, which effectively overcomes the inhibition of yeast growth caused by the low C / N ratio of the original biogas slurry. It significantly promotes the biomass accumulation of strain Delburo's Flocculated Yeast L12345 in the biogas slurry, and transforms the "waste" biogas slurry into a "culture medium" suitable for bacterial growth.

[0024] 3. Highly efficient synergistic effect of pollution removal and resource recovery: This method effectively removes ammonia nitrogen and COD from biogas slurry while producing high-value single-cell protein, achieving "treating waste with waste and turning waste into treasure". Under optimized conditions, the ammonia nitrogen removal rate can reach 44.3%, the COD removal rate can reach 27.3%, and the SCP yield can reach 7.13 g / L.

[0025] 4. Low cost and significant economic benefits: The main raw materials, biogas slurry and molasses, are inexpensive industrial and agricultural by-products or wastes, which greatly reduces production costs. Calculations show that this process can generate a net profit of approximately RMB 130.5 per cubic meter of biogas slurry treated. Compared to traditional processes that only meet emission standards or involve partial resource recovery, this process offers significant economic benefits and has great application and promotion value.

[0026] 5. Mild operating conditions and environmentally friendly: The entire process is a biological fermentation process, which does not require the addition of expensive chemical agents. It is carried out at normal pressure and medium temperature, with low energy consumption and no secondary pollution, which meets the requirements of green and sustainable development. Attached Figure Description

[0027] Figure 1 This is a flowchart illustrating the screening and identification process of Torulaspora delbrueckii L12345 in an embodiment of the present invention.

[0028] Figure 2 This is a colony morphology diagram of *Cytomyces delbrueckii* L12345 screened in an embodiment of the present invention.

[0029] Figure 3 This is a microscopic (×1000) observation image of the *Cytomyces delbrueckii* L12345 strain selected in this embodiment of the invention.

[0030] Figure 4 In this embodiment of the invention, the strain *Cytomyces delbrueckii* L12345 was amplified and sequenced using ITS primers, and a phylogenetic tree was constructed by comparing and selecting gene sequences with high homology using NCBI's Blast sequence analysis.

[0031] Figure 5 This is a growth diagram of strain Delbuspira spores L12345 under different ammonia nitrogen concentrations (C / N fixed) in an embodiment of the present invention.

[0032] Figure 6 This is a process flow diagram of the production of single-cell protein from biogas slurry using strain Delbrio parahaemolyticus L12345 in an embodiment of the present invention.

[0033] Figure 7 This is a graph showing the effect of different cultivation times on SCP yield and pollutant removal rate in an embodiment of the present invention.

[0034] Figure 8 This is a graph showing the effect of different initial pH values ​​on SCP yield and pollutant removal rate in an embodiment of the present invention.

[0035] Figure 9 This is a graph showing the effect of different inoculation ratios on SCP yield and pollutant removal rate in embodiments of the present invention.

[0036] Figure 10 This is a graph showing the effect of different C / N ratios on SCP yield and pollutant removal rate in embodiments of the present invention.

[0037] Figure 11 A comparison chart showing the effect of adding molasses on the resource utilization of biogas slurry by *Cytomyces delbrueckii* L12345. Detailed Implementation

[0038] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0039] It is understood that, unless otherwise specified, the raw materials and operations used in each embodiment can be achieved using conventional experimental materials and methods.

[0040] Example 1: Screening and identification of high ammonia nitrogen tolerant yeast strain Torulaspora delbrueckii L12345

[0041] The main process is as follows Figure 1 As shown, the main steps include:

[0042] (1) Sample collection and enrichment: biogas slurry was collected from a biogas station in Xuzhou, and wastewater from pig farms and lees from a distillery were collected as screening sources.

[0043] (2) Initial screening: The supernatant of each sample was serially diluted and spread on yeast extract peptone glucose agar (YPD) plates and incubated at 30 ℃ for 5 days. Single colonies with different morphologies were picked for purification culture.

[0044] (3) High ammonia nitrogen tolerance screening: The purified strains were inoculated on a solid selective medium with (NH4)2SO4 as the sole nitrogen source and an ammonia nitrogen concentration of 3000 mg / L. They were cultured at 30°C for 5 days, and strains with vigorous growth and large colony diameter were selected as the primary screening strains tolerant to high ammonia nitrogen.

[0045] (4) Secondary screening: The strains screened in the primary screening were inoculated into shake flasks with actual biogas slurry (ammonia nitrogen concentration of approximately 2930 mg / L) as the culture medium and cultured at 30°C and 180 rpm for 5 days. The changes in ammonia nitrogen and COD of the biogas slurry before and after culture were measured, and the bacterial cells were collected, dried, and weighed to calculate the SCP yield. Finally, a strain that grew relatively best in biogas slurry and had the strongest ammonia nitrogen removal capacity was selected and numbered L12345.

[0046] (5) Identification: Morphological observation and ITS rDNA sequence analysis were performed on strain L12345. Morphologically, the colonies were milky white with a smooth, raised surface; the cells were oval or elliptical, such as... Figure 2 and Figure 3 As shown. Molecular identification results indicate that this strain has the highest homology with Torulaspora delbrueckii, such as... Figure 4 As shown, it was therefore identified as Torulaspora delbrueckii.

[0047] Example 2: Ammonia nitrogen tolerance test of *Cyclophorus delbrueckii* L12345

[0048] Liquid selective media with different ammonia nitrogen concentrations (1000 mg / L, 2000 mg / L, 3000 mg / L, 4000 mg / L, 5000 mg / L) were prepared, maintaining a C / N ratio of 3. Seed culture of *Cyclocarya delbue* L12345 in the logarithmic growth phase was inoculated into the above media and cultured at 30°C with shaking at 180 rpm for 5 days. The final OD was then measured. 600 The value is used to assess growth.

[0049] The results are as follows Figure 5 As shown, strain *Cyclocarya delbue* L12345 maintained good growth (OD) even at ammonia nitrogen concentrations as high as 4000 mg / L. 600 The ammonia nitrogen concentration was >0.9 mg / L, but growth was severely inhibited when the concentration reached 5000 mg / L. This indicates that strain *Cyclocarya delbue* L12345 exhibits extremely strong tolerance to high ammonia nitrogen environments.

[0050] Example 3: Process optimization for producing SCP from biogas slurry using strain *Cyclophorus delbrueckii* L12345.

[0051] The process for producing SCP using biogas slurry treated with strain Delbrio spores L12345 is as follows: Figure 6 As shown, the main steps include:

[0052] S1. Pretreatment of biogas slurry: Collect biogas slurry produced by biogas projects, let it stand or centrifuge to remove large suspended particles, and obtain pretreated biogas slurry.

[0053] S2. Culture medium preparation: Add sugarcane molasses as an exogenous carbon source to the pretreated biogas slurry, stir and mix evenly to prepare a biogas slurry-molasses mixed culture medium; adjust the carbon-nitrogen ratio (C / N) of the mixed culture medium to 3.0~15.0 by adding the molasses.

[0054] S3. Culture medium conditioning and sterilization: Adjust the initial pH of the mixed culture medium prepared in step S2 to 4.0~9.0, and then sterilize it.

[0055] S4. Inoculation and culture: The seed culture of *Cytomyces delbrueckii* L12345 in the logarithmic growth phase was inoculated into the sterilized mixed culture medium at a volume ratio of 5% to 30%. The culture was carried out aerobically at a temperature of 30 ℃ and a shaking speed of 180 rpm for 1 to 5 days.

[0056] S5. Product Harvesting: After the culture is completed, the fermentation broth is separated into solid and liquid components, and the bacterial biomass is collected. After washing and drying, the single-cell protein product is obtained.

[0057] Pretreated biogas slurry (ammonia nitrogen 2930 mg / L, TOC 4.937 g / L) was used as the basal culture medium, with sugarcane molasses added to adjust the C / N ratio. In single-factor experiments, the effects of culture time (1–5 days), initial pH (4.0–9.0), inoculum ratio (5%–30%), and C / N ratio (3.0–15.0) on SCP yield, ammonia nitrogen removal rate, and COD removal rate were investigated. Basic culture conditions were: temperature 30℃, shaker speed 180 rpm.

[0058] Incubation time: such as Figure 7 As shown, SCP yield, ammonia nitrogen and COD removal rate increased rapidly in the first 3 days of cultivation, reached their peak on the 4th day and then tended to stabilize. Therefore, the optimal cultivation time is 4 days.

[0059] Initial pH: as Figure 8 As shown, within the pH range of 4.0–6.0, SCP production increases with increasing pH, peaking at pH 6.0; however, SCP production decreases sharply after pH > 7.0. Ammonia nitrogen removal rate continuously decreases with increasing pH, which is related to the increased proportion of free ammonia. The optimal initial pH is 6.0.

[0060] Vaccination ratio: such as Figure 9 As shown, when the vaccination rate is between 5% and 20%, all indicators increase slowly with the increase in the vaccination rate; however, the improvement is not significant after the vaccination rate exceeds 20%. From a cost-efficiency perspective, the optimal vaccination rate is 20%.

[0061] C / N ratio: such as Figure 10 As shown, SCP production reaches its peak at a C / N ratio of 9; excessively low or high C / N ratios are detrimental to production growth. Ammonia nitrogen removal rate decreases with increasing C / N ratio, while COD removal rate declines due to the additional COD introduced by molasses. The optimal C / N ratio is determined to be 9.0.

[0062] Example 4: Treatment effect under optimal process conditions

[0063] Based on the optimization results of Example 3, a verification experiment was conducted under the following optimal conditions: biogas slurry-molasses mixed culture medium, culture time of 4 days, initial pH 6.0, C / N ratio of 9.0, inoculum ratio of 20%, temperature of 30 ℃, and rotation speed of 180 rpm. The treatment results are shown in Table 1.

[0064] Table 1. Effect of Derbus spore-forming yeast L12345 on biogas slurry under optimal conditions.

[0065] Under these conditions, strain Delbuchondrodysporum L12345 efficiently converts pollutants in biogas slurry into high-quality microbial protein, significantly increasing SCP production and exhibiting rich microbial protein content, thus possessing high feed application value.

[0066] Example 5: Preliminary Analysis of Process Economics

[0067] Based on the treatment of 1 cubic meter of biogas slurry:

[0068] Costs: mainly include consumables for preparing seed solution, equipment operation and labor costs, which are initially estimated at about 122.6 yuan.

[0069] Revenue: The main product is dried bacterial culture (SCP), with a yield of approximately 7.13 kg. Based on the market price of similar microbial proteins (approximately US$5 / kg, equivalent to RMB 35 / kg), the output value is approximately RMB 253.1.

[0070] Net profit: After deducting costs from output value, approximately RMB 130.5 can be obtained for each cubic meter of biogas slurry processed.

[0071] Compared to traditional biogas slurry discharge standards (costing approximately 150-200 yuan / ton with no profit) or partial resource recovery processes, the process of this invention exhibits significant economic advantages.

[0072] Comparative example: Treatment of raw biogas slurry without the addition of molasses

[0073] Without adding molasses (i.e., maintaining the original C / N ratio of the biogas slurry, approximately 1.7), after 5 days of cultivation, the SCP yield was only 0.24 g / L, the ammonia nitrogen removal rate was 30.5%, and the COD removal rate was 9.4%. Figure 11 As shown, the results are significantly lower than those of Example 4 compared to the optimized process of this invention, which fully demonstrates the key role of adding inexpensive carbon source (molasses) to adjust the C / N ratio in overcoming the inherent defects of biogas slurry and activating the production potential of strain Delburo's spore-forming yeast L12345.

[0074] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A high-ammonia nitrogen-tolerant yeast strain, characterized in that, The strain, named *Torulaspora delbrueckii* L12345, was deposited on January 13, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 39123. The OD500 of *Torulaspora delbrueckii* L12345 at an ammonia nitrogen concentration of 4000 mg / L was measured. 600 >0.

9.

2. The application of the high ammonia nitrogen-tolerant yeast of claim 1 in the resource utilization treatment of biogas slurry, the application comprising inoculating *Cyclophorus delbrueckii* L12345 into a culture medium with high ammonia nitrogen biogas slurry as the main substrate for fermentation culture to obtain single-cell protein products, wherein the ammonia nitrogen concentration of the high ammonia nitrogen biogas slurry is 3000 mg / L, the culture medium is a biogas slurry-molasses mixed culture medium with added molasses, the carbon-nitrogen ratio of the biogas slurry-molasses mixed culture medium is 9.0, the initial pH value is 6.0, and the fermentation culture is carried out aerobically for 4 days at a temperature of 30 ℃ and a shaking speed of 180 rpm.

3. A method for producing single-cell protein by treating biogas slurry using the high-ammonia-nitrogen-tolerant yeast of claim 1, characterized in that, Includes the following steps: S1. Pretreatment of biogas slurry: Collect biogas slurry and remove large particulate suspended matter to obtain pretreated biogas slurry with an ammonia nitrogen concentration of 3000 mg / L. S2. Preparation of biogas slurry-molasses mixed culture medium: Add molasses to the pretreated biogas slurry, adjust the carbon-nitrogen ratio of the biogas slurry-molasses mixed culture medium to 9.0, adjust the initial pH of the mixed culture medium to 6.0, and then sterilize it. S3. Inoculation and culture: The seed culture of *Cytomyces delbrueckii* L12345 in the logarithmic growth phase was inoculated into sterilized biogas slurry-molasses mixed medium at a volume ratio of 20%. It was then cultured aerobically at 30 ℃ and 180 rpm for 4 days. S4. Product Harvesting: After the culture is completed, the fermentation broth is separated into solid and liquid components, and the bacterial biomass is collected. After washing and drying, the single-cell protein product is obtained.

4. The method according to claim 3, characterized in that, The molasses mentioned in step S2 is sugarcane molasses.

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