High-protein chlorella and application thereof in resourceful treatment of high free ammonia wastewater

Abstract

The present application relates to a kind of high protein chlorella and its application in high free ammonia wastewater resource treatment, the chlorella is screened from biogas liquid and obtained chlorella SXU-03, preservation number is CCTCC NO:M 2026669.The crude protein content of the strain is 56.15±0.19%, essential amino acid is rich and balanced, and is rich in limiting amino acid such as lysine, with the nutritional basis as high-quality feed protein source.The present application inoculates the strain in high free ammonia wastewater and carries out compatible culture, can tolerate initial concentration up to 490 mg / L free ammonia stress and keep proliferation.The present application overcomes the bottleneck of high cost in industrial amplification in traditional water dilution or high-speed centrifugal pretreatment, effectively degrades wastewater ammonia nitrogen load, while realizing the synchronous accumulation of high-value microalgae protein biomass, provides extremely economic and feasible industrial implementation plan for resource treatment of high ammonia nitrogen wastewater.

Description

Technical Field

[0001] This invention belongs to the field of microbial and environmental engineering technology, and specifically relates to a high-protein Chlorella and its application in the resource-based treatment of high-free ammonia wastewater. Background Technology

[0002] With the rapid development of large-scale livestock and poultry farming, biogas slurry produced by anaerobic fermentation has become an important usable resource due to its rich content of nutrients such as nitrogen and phosphorus. Utilizing microalgae to treat biogas slurry not only achieves deep nitrogen and phosphorus removal from the wastewater but is also a core pathway to realize the resource utilization of waste. Currently, livestock and poultry farming heavily relies on traditional high-priced protein feeds such as soybean meal and fishmeal, and conventional plant-based feeds (such as corn) generally suffer from severe nutritional deficiencies in essential amino acids such as lysine and methionine. High-protein microalgal biomass not only has a high crude protein content but also a balanced composition of essential amino acids, making it an extremely high-quality new feed protein source. Applying high-protein algae strains to biogas slurry treatment can directly transform high-concentration pollution loads into high-value-added biological resources that compensate for the shortcomings of traditional feeds, perfectly aligning with the resource utilization trend of "treating waste with waste and creating value with algae" in modern environmental protection engineering.

[0003] However, in practical engineering applications, the high ammonia nitrogen and alkaline environment of livestock and poultry biogas slurry produces large amounts of highly toxic free ammonia. Free ammonia easily penetrates the cell membranes of microalgae, triggering severe oxidative stress and DNA damage, leading to a precipitous drop in photosynthetic efficiency. Numerous studies have shown that common microalgae (such as Chlorella proteoglycans and Chlorella sorokinense) have extremely low tolerance limits to free ammonia: significant growth inhibition typically occurs at 30-40 mg / L, and when the concentration exceeds 75-120 mg / L, the photosynthetic system is severely damaged, essentially halting proliferation or even causing death. Although a few native selected strains have slightly higher tolerance thresholds (115-153 mg / L), they still cannot effectively survive and proliferate when faced with real livestock and poultry biogas slurry with free ammonia concentrations often reaching hundreds of mg / L.

[0004] To reduce the toxicity of free ammonia, existing technologies often employ idealized laboratory methods such as high-ratio dilution with clean water or high-speed centrifugation for pretreatment of biogas slurry. However, in the actual large-scale operation of wastewater treatment plants, these methods not only significantly increase water consumption, but their high equipment and energy costs also completely contradict the economic feasibility of industrial-scale implementation. Summary of the Invention

[0005] The purpose of this invention is to provide a high-protein Chlorella and its application in the resource utilization treatment of high free ammonia wastewater. This algal strain has a high protein content, excellent tolerance to high free ammonia, high efficiency in nutrient conversion, and high efficiency in the purification treatment of high free ammonia wastewater.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A high-protein Chlorella species and its application in the resource utilization treatment of high-free ammonia wastewater, wherein the Chlorella strain is Chlorella sorokiniana SXU-03, deposited on April 13, 2026, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 2026669.

[0008] This invention also provides the application of the above-mentioned high-protein Chlorella in the resource utilization treatment of high-free ammonia wastewater.

[0009] The present invention also provides the application of the above-described high-protein Chlorella in the preparation of animal feed.

[0010] The present invention also provides a method for treating high-free ammonia wastewater with the above-mentioned high-protein Chlorella, comprising the following steps: inoculating the culture medium of high-protein Chlorella into the high-free ammonia wastewater, and adding an organic carbon source for the co-culture of algae.

[0011] Preferably, the initial free ammonia concentration of the high-free ammonia wastewater is less than 490 mg / L, and the pH value is 7.5-9.0.

[0012] Preferably, the inoculum amount of the high-protein Chlorella is 0.5 g·L⁻¹ of dry weight of the algae, and the organic carbon source is glucose, with a glucose addition amount of 2.5 g·L⁻¹.

[0013] Preferably, the conditions for the mixed culture are: temperature of 26.5±0.5℃, light intensity of 3000 lux, photoperiod of light-dark ratio of 12:12, and continuous shaking culture at 150 rpm for 7 days.

[0014] Preferably, before inoculating the high-free-ammonia wastewater, the high-protein Chlorella is first cultured autotrophically in BG11 liquid medium until the logarithmic growth phase.

[0015] Preferably, the autotrophic culture environment conditions in the BG11 liquid culture medium are: temperature 22.0-27.0℃, pH 6.8-7.6, and shaking the culture container 2-3 times a day; the light conditions are: light intensity 3000 lux, and photoperiod light-dark ratio 12:12.

[0016] This invention first screened and obtained a strain of *Chlorella sorokinense* with the preservation number CCTCC NO: M 2026669. Quantitative analysis showed that its crude protein content was as high as 56.15±0.19%, and its essential amino acid composition was rich and balanced (ΣEAA reached 23.26%), meeting the standards for high-quality feed protein sources and establishing its extremely high resource utilization value as a novel microalgal protein source. Based on this high protein characteristic, further studies on free ammonia stress showed that this algal strain exhibited excellent ammonia nitrogen tolerance under multi-trophic culture conditions. The preserved algal strain of this invention could withstand extreme free ammonia stress of up to 490 mg / L in the treatment of high-free ammonia wastewater. Standard algal strains isolated from conventional environments often suffer photosynthetic system damage or even death in such high-free ammonia environments, while the algal strain of this invention can directly survive and maintain proliferation in extreme water quality without relying on economically infeasible industrial pretreatment methods such as high-ratio dilution with clean water or high-speed centrifugation. This algal strain perfectly combines the dual advantages of "high protein endowment" and "extremely strong tolerance to high free ammonia," enabling it not only to effectively overcome the toxicity inhibition of high free ammonia wastewater and achieve purification, but also to efficiently proliferate and accumulate high-value microalgal protein biomass in harsh water quality environments, truly achieving the dual goals of effective purification of high free ammonia wastewater and development of high-value-added feed protein raw materials. Attached Figure Description

[0017] Figure 1 The cell morphology of the high-protein Chlorella strain of this invention under an optical microscope (10x eyepiece × 100x objective).

[0018] Figure 2 A phylogenetic tree was constructed using BioEdit 7.2 to analyze the partial gene sequences of the high-protein Chlorella strain of this invention, based on the combined 18S and ITS sequences.

[0019] Figure 3 This is a comparison of the biomass of the high-protein Chlorella strain of this invention in high ammonia nitrogen wastewater over 7 days. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0021] The specific steps for collecting, separating, and purifying the high-protein Chlorella of this invention are as follows:

[0022] (1) Sample collection:

[0023] The high-protein Chlorella described in this invention was collected from the biogas slurry pond of the cattle farm of Shanxi Huaxin Biomass Energy Development Co., Ltd. in Ying County, Shanxi Province. Water samples were collected from selected areas, placed in 50mL sampling bottles, and stored in a refrigerator at 4°C.

[0024] (2) Isolation, purification and culture of algal strains:

[0025] Algal strains were isolated using a combination of dilution separation and solid culture medium. Water samples were filtered through 200-300 mesh gauze and then added to Erlenmeyer flasks containing BG11 liquid medium (BG11 formulation shown in Table 1). The algal strains were enriched and cultured in a light incubator. After 5-7 days, the medium changed from colorless to light green. The medium was then evenly spread onto prepared, autoclaved BG11 solid medium (containing 2-3% agar powder, with ammonium chloride as the nitrogen source, and an initial pH of 8.5) using a spreader. The high concentration of free ammonia carried by the BG11 solid medium was used to screen for tolerant algal strains. The uniformity of cell morphology was then observed using an optical microscope. If the morphology was uniform, the separation was successful; otherwise, the plate spreading process was repeated on the solid medium until uniform cell morphology was observed. The algal strains were inoculated into Erlenmeyer flasks containing liquid BG11 medium using an inoculation loop and placed in a light incubator for shaking culture until green colonies appeared in the medium.

[0026] The algal strains cultured in the light incubator were expanded in 250mL Erlenmeyer flasks (effective culture volume of 100mL), using BG11 medium, at a temperature of 25.0±0.5℃, a pH of 7.0, a light intensity of 3000 lux, and a photoperiod of 12:12 light-dark ratio.

[0027] Table 1. BG11 Formulation and Dosage

[0028]

[0029] Note: After the culture medium is prepared, use 1 mol·L⁻¹ -1 HCl and 1 mol·L -1 The pH was adjusted to 7.0-7.2 with NaOH, and then sterilized at 121℃ for 30 minutes in an autoclave.

[0030] Table 2 A5 Formula

[0031]

[0032] The identification of the algal strain is completed in two steps: first, a preliminary observation of cell morphology is performed, followed by molecular biological identification. The morphology, size, structure, and other characteristics of the isolated and purified algal strain are observed and photographed using an optical microscope. The algal strain described in this invention is as follows... Figure 1 As shown.

[0033] The algal strains isolated and purified in this invention, when observed under an optical microscope, are green in color, with unicellular ellipsoidal bodies and a few multicellular colonies containing nuclei. Molecular biological identification of the algal strains was performed using extracted DNA as a template. Primers for 18S rRNA and ITS were selected from universal primers for green algae; the primer sequences are shown in Table 3. PCR amplification of the 18S rRNA and ITS genes was performed, establishing a PCR reaction system. The PCR reaction system and operation procedure are shown in Tables 4 and 5. After the reaction, 5 μL of the PCR product was subjected to agarose gel electrophoresis, and positive clones were then screened. Sequencing was performed by Beijing Liuhe Huada Genomics Co., Ltd. Unreliable sequences at both ends were removed from the sequencing results. The obtained sequences were then compared with those in the GenBank database using Nucleotide BLAST for homology detection, and compared with existing algal gene sequences in the algal gene bank to finally determine the species.

[0034] After PCR amplification of the algal strain, 18S rRNA and ITS fragments were obtained. Sequencing revealed that the lengths of the 18S rRNA and ITS fragments were 1779 bp and 743 bp, respectively. A phylogenetic tree of the Chlorellaceae family was constructed based on the 18S rDNA and ITS sequences, revealing that it is most closely related to *Chlorella sorokinense*. The results are as follows: Figure 2 As shown.

[0035] The *Chlorella sorokinica* strain obtained and identified through isolation and purification was deposited at the China Center for Type Culture Collection (CCTCC) (Wuhan University, Wuhan, China) on April 13, 2026, with accession number: CCTCC NO: M 2026669.

[0036] Table 3 Primer sequences for the target gene

[0037]

[0038] Table 4 PCR reaction system

[0039]

[0040] Note: The DNA concentration in the normal system should be 30-50 ng / mL. If the DNA concentration is too low (10-15 ng / mL), increase the DNA to 6 μL (2x3). Correspondingly, reduce the amount of ddH2O to 5.8 μL and maintain a reaction system of 20 μL.

[0041] Table 5 PCR Operation Procedure

[0042]

[0043] Note: A total of 35 cycles were performed. After the reaction was completed, the product was stored at 4°C.

[0044] Determination of protein and amino acid content in high-protein Chlorella strain CCTCC NO: M 2026669

[0045] In this embodiment, the crude protein content of high-protein Chlorella sorokiniana (SXU-03, preservation number: CCTCC NO: M 2026669) was determined using the Kjeldahl method. The specific steps are as follows:

[0046] Protein content determination

[0047] (1) Sample preparation: collect microalgae from the culture system by centrifugation, wash them 2-3 times with deionized water, freeze-dry them under vacuum, and grind them into uniform algal powder for later use.

[0048] (2) Sample digestion: Accurately weigh 0.5 g of freeze-dried algal powder and place it in a Kjeldahl nitrogen determination digestion tube. Add 0.5 g of anhydrous copper sulfate and 4.5 g of potassium sulfate mixed catalyst and 10 mL of concentrated sulfuric acid. Heat the sample in a digestion furnace in stages: first heat to 280℃ and hold for 20 min, then heat to 400℃ and hold for 40 min, and cool to room temperature before testing.

[0049] (3) Distillation and titration: Connect the cooled digestion tube to an automated Kjeldahl nitrogen analyzer, automatically add excess concentrated sodium hydroxide solution (50 mL) for alkalization, and perform steam distillation (90% steam flow rate). The released ammonia gas is introduced into boric acid absorption solution (30 mL) containing methyl red-bromocresol green indicator. Distill for 5 min, and then titrate with a standard quantitative hydrochloric acid solution to the endpoint (from blue-green to purple-red), and record the volume consumed. Perform a blank control experiment in the same way.

[0050] (4) Content calculation: The total nitrogen content of the sample was calculated to be 8.98±0.03% based on the consumption of standard hydrochloric acid solution. Multiplying by the microalgae crude protein conversion factor of 6.25, the crude protein content of the high-protein Chlorella described in this invention is 56.15±0.19%.

[0051] Determination of amino acid composition and content

[0052] The amino acid composition of high-protein Chlorella was determined using a fully automated amino acid analyzer. The specific steps are as follows:

[0053] Accurately weigh 1g of freeze-dried and uniformly ground microalgae powder, place it in a glass hydrolysis tube, add 10 mL of 6mol / L hydrochloric acid solution and 3-4 drops of phenol. Freeze the hydrolysis tube under vacuum, purge it with nitrogen, and seal it. Place it in a 110℃ incubator for continuous hydrolysis for 24 hours. After hydrolysis, cool and open the tube, filter the hydrolysate, and transfer it to a rotary evaporator. Remove the hydrochloric acid by rotary evaporation under reduced pressure at 55℃. Then, bring the volume of the residue to 25 mL using pH 2.2 sodium citrate buffer. Filter the solution through a 0.22μm microporous membrane, and use an automated amino acid analyzer for post-column ninhydrin derivatization and colorimetric analysis. Calculate the content of each amino acid component by comparing its retention time and peak area with 17 amino acid standards.

[0054] (2) Measurement results and comparative analysis

[0055] As shown in Table 6, the *Chlorella sorokinense* SXU-03 described in this invention exhibits extremely superior amino acid composition characteristics, and its nutritional value for feed is significantly better than that of conventional feed ingredients and conventional algae strains. Specifically, the total essential amino acid (ΣEAA) of the algae strain of this invention is as high as 23.26%, which is not only significantly better than conventional *Chlorella* (19.99%), but also far exceeds that of high-quality plant protein source soybean meal (19.26%) and basic energy feed corn kernels (3.31%). In particular, regarding the key limiting amino acids most easily lacking in conventional livestock and poultry diets, the lysine (Lys) and methionine (Met) contents of the algae strain of this invention are as high as 4.75% and 1.18%, respectively, surpassing conventional *Chlorella* (Lys 2.86%, Met 0.92%), and significantly leading soybean meal (Lys 2.85%, Met 0.45%) and corn (Lys 0.46%, Met 0.10%).

[0056] Table 6. Amino acid composition and content

[0057]

[0058] Note: ΣEAA represents the total amount of essential amino acids, ΣNEAA represents the total amount of non-essential amino acids, and ΣAA represents the total amount of amino acids. The amino acid composition and content of corn kernels are referenced in [1] Zheng Jingui, Tu Jiefeng, Chen Junchen, et al. Study on protein content and amino acid composition of several plant feed resources [J]. Journal of Fujian Academy of Agricultural Sciences, 1991, (2): 39-44; The amino acid composition and content of soybean meal and Chlorella are referenced in [2] Tang Li, Jia Luyao, Sun Jian, et al. Effects of Chlorella replacing soybean meal on growth performance, muscle quality and intestinal flora of Mirror Carp [J]. Acta Hydrobiologica Sinica, 2026, 1-20.

[0059] Investigation into the tolerance of high-protein Chlorella strain CCTCC NO: M 2026669 to free ammonia conditions

[0060] (1) Preparation of artificially synthesized high-free-ammonia wastewater:

[0061] To eliminate interference from complex matrices in real wastewater and accurately assess the stress effect of single free ammonia on microalgae, this embodiment uses artificially synthesized high-free-ammonia wastewater for the experiment. The specific formulation of the wastewater is shown in Table 7.

[0062] Table 7 Wastewater formulation and dosage

[0063]

[0064] Note: After the culture medium is prepared, use 1 mol·L⁻¹ -1 HCl and 1 mol·L -1 The pH was adjusted to 7.5-9.0 with NaOH, and the mixture was sterilized at 110℃ for 15 minutes in an autoclave. The A5 formula is shown in Table 2.

[0065] (2) Construction and cultivation of different free ammonia concentration gradients:

[0066] In the prepared synthetic wastewater, ammonium chloride was used as the main nitrogen source, and 1 mol·L⁻¹ was added. -1 HCl and 1 mol·L -1 The initial pH of the wastewater was precisely controlled using NaOH, thus creating four gradient groups with different initial free ammonia concentrations:

[0067] Group 1: Adjust the pH to 7.5, corresponding to an initial free ammonia concentration of 30 mg / L;

[0068] Group 2: Adjust the pH to 8.0, corresponding to an initial free ammonia concentration of 90 mg / L;

[0069] Group 3: Adjust the pH to 8.5, corresponding to an initial free ammonia concentration of 243 mg / L;

[0070] Group 4: Adjust the pH to 9.0, corresponding to an initial free ammonia concentration of 490 mg / L.

[0071] High-protein Chlorella in the logarithmic growth phase was measured at a dry weight of 0.5 g·L⁻¹. -1 The inoculum amounts were respectively added to the four groups of wastewater. The inoculum was then placed in a light-shaking incubator for multi-trophic cultivation. The cultivation conditions were set as follows: temperature 26.5±0.5℃, light intensity 3000 lux, photoperiod light-dark ratio 12:12, and continuous shaking at 150 rpm for 7 days. Daily samples were taken to measure changes in microalgal biomass (g / L) and free ammonia concentration (mg / L) in the system.

[0072] (3) Experimental results and analysis:

[0073] The growth of high-protein Chlorella in wastewater with different concentrations of free ammonia is as follows: Figure 3 As shown in the figure. This preserved algal strain showed a strong ability to adapt to and recover from free ammonia in the co-trophic mode. In the low free ammonia stress groups (pH 7.5 and pH 8.0, initial FA ≤ 90 mg / L), the microalgae had almost no obvious growth cessation period. The pH 8.0 group reached the highest biomass on the 7th day of culture.

[0074] In the extremely high free ammonia stress groups (pH 8.5 and pH 9.0, initial FA 243 mg / L and 490 mg / L, respectively), the microalgae experienced acute toxicity inhibition from free ammonia during the first 1-2 days of cultivation, resulting in a slight decrease in biomass. However, due to the physiological protective barrier provided by the organic carbon source of 2.5 g / L glucose in the formulation, the microalgae began to grow slowly on the 3rd day. Subsequently, the biomass of the microalgae in the high-stress group began to steadily increase, demonstrating that this algal strain could withstand extreme free ammonia shocks up to 490 mg / L and maintain its proliferative capacity. Meanwhile, comparing the growth changes of several Chlorella strains from different sources reported in the literature under free ammonia stress (Table 8), significant growth inhibition usually occurred at 30-40 mg / L, and when the concentration exceeded 75-120 mg / L, the photosynthetic system was severely disrupted, essentially halting proliferation or even leading to death. The *Chlorella sorokinense* SXU-03 of this invention exhibited a significantly enhanced tolerance. Under extreme free ammonia shocks of up to 490 mg / L, this algal strain not only avoided lethal damage but also achieved steady biomass accumulation through metabolic regulation in a cotrophic mode.

[0075] Table 8. Growth changes of Chlorella from different sources under free ammonia stress

[0076]

[0077] The above embodiments confirm that the preserved algal strain screened and verified by this invention is a high-quality, high-protein microalga with a crude protein content as high as 56.15±0.19%. In a co-trophic mode with the addition of an organic carbon source, its tolerance limit for free ammonia concentration can reach 490 mg / L. This characteristic makes it fully capable of directly surviving in undiluted livestock biogas slurry with high ammonia nitrogen and high pH, ​​degrading ammonia nitrogen, and efficiently accumulating biomass for industrial application.

Claims

1. A high-protein Chlorella, characterized in that, The Chlorella strain mentioned is Chlorella sorokinina SXU-03, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026669.

2. The application of the high-protein Chlorella described in claim 1 in the resource utilization treatment of high-free ammonia wastewater.

3. The application of the high-protein Chlorella described in claim 1 in the preparation of animal feed.

4. A method for treating high-free-ammonia wastewater using Chlorella vulgaris as described in claim 1, characterized in that, Includes the following steps: The culture medium of high-protein Chlorella was inoculated into wastewater with high free ammonia, and an organic carbon source was added for the ditrophic culture of the algae.

5. The application according to claim 2 or the method according to claim 4, characterized in that, The initial free ammonia concentration of the high-free ammonia wastewater is less than 490 mg / L, and the pH value is 7.5-9.

0.

6. The method according to claim 4, characterized in that, The inoculation amount of the high-protein Chlorella is 0.5-0.55 g·L⁻¹ of the dry weight of the algal strain, and the organic carbon source is glucose, with a glucose addition amount of 2.5 g·L⁻¹.

7. The method according to claim 4, characterized in that, The conditions for the mixed culture were: temperature 26.5±0.5℃, light intensity 3000-3500 lux, photoperiod light-dark ratio 12:12, and continuous shaking culture at 150 rpm.

8. The method according to claim 4, characterized in that, Before inoculating the high-free-ammonia wastewater, the high-protein Chlorella was first cultured autotrophically in BG11 liquid medium until the logarithmic growth phase.

9. The method according to claim 7, characterized in that, The autotrophic culture environment conditions in the BG11 liquid medium are: temperature 22.0-27.0℃, pH 6.8-7.6, and shaking the culture container 2-3 times a day; the light conditions are: light intensity 3000 lux and photoperiod light-dark ratio 12:12.

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