A low-temperature resistant spirulina and its application in the treatment of livestock and poultry wastewater in cold regions

Abstract

This invention relates to the field of microalgae technology, specifically providing a low-temperature resistant spirulina and its application in the treatment of livestock and poultry wastewater in cold regions. The spirulina provided by this invention has the preservation number CGMCC No. 40697. The spirulina provided by this invention exhibits good growth under both normal and low temperatures, in culture media or biogas slurry environments. The low-temperature resistant spirulina provided by this invention also demonstrates tolerance to high concentrations of ammonia nitrogen and phosphorus, providing a green and safe method for treating high-ammonia-nitrogen and high-phosphorus livestock and poultry wastewater in cold regions.

Description

Technical Field

[0001] This invention relates to the field of microalgae technology, specifically providing a low-temperature resistant spirulina and its application in the treatment of livestock and poultry wastewater in cold regions. Background Technology

[0002] The development of livestock farming generates a large amount of livestock manure. Treating livestock waste through large-scale biogas fermentation using this manure as raw material can both control pollution and achieve resource utilization of waste, yielding triple benefits in terms of energy, environmental protection, and the economy, aligning with the concept of sustainable development for human society. However, the biogas slurry produced by large-scale biogas projects, having only undergone anaerobic treatment, still contains large amounts of nutrients such as nitrogen and phosphorus. Direct discharge into water bodies can easily cause eutrophication and secondary pollution, while also placing significant pressure on local disposal.

[0003] Microalgae are autotrophic organisms with a strong ability to convert nitrogen and phosphorus. They can absorb nutrients such as carbon, nitrogen, and phosphorus from wastewater (biogas slurry) to promote their own growth, thus purifying the wastewater in the process. The harvested algal cell biomass can then be used as microalgal biofuel, feed additives, or other high-value-added products, representing a green and low-cost industrial development approach for microalgae cultivation. Therefore, utilizing microalgae to treat wastewater can remove pollutants, fix carbon, and produce high-value products simultaneously, making it a sustainable wastewater treatment technology with broad development prospects.

[0004] Large-scale biogas projects in cold regions produce biogas slurry rich in nutrients such as nitrogen and phosphorus, which need to be recovered. Microalgae, as autotrophic organisms, can absorb CO2 from the air under light and simultaneously utilize nitrogen and phosphorus nutrients in wastewater to synthesize their own algal bodies, exhibiting a strong nitrogen and phosphorus conversion capacity. However, the low-temperature environment in cold regions leads to problems such as poor microbial activity, slow reaction, and low material conversion efficiency during the treatment process, severely restricting the efficiency of livestock and poultry wastewater treatment and resource utilization. Therefore, how to conduct low-cost, in-depth on-site treatment of livestock and poultry wastewater, especially research on the resource utilization of carbon, nitrogen, and phosphorus elements under the low-temperature effect, is one of the main problems to be solved in the resource utilization treatment of poultry wastewater in cold regions.

[0005] For the treatment of livestock and poultry wastewater in cold regions, existing technologies do not have microalgae strains that are well-tolerant to specific biogas slurries and grow well in cold regions. Summary of the Invention

[0006] This invention provides a low-temperature resistant spirulina and its application in the treatment of livestock and poultry wastewater in cold regions.

[0007] The technical problems encountered in using microalgae to treat livestock and poultry wastewater in cold regions are as follows: (1) High concentrations of ammonia nitrogen in livestock and poultry wastewater inhibit the growth of microalgae. Furthermore, the current concentration of biogas slurry used to cultivate microalgae in pig manure biogas slurry is 60-600 mg / L. There are currently no low-temperature algae species (5-15℃) that can grow in environments with biogas slurry concentrations higher than 400 mg / L and have good ammonia nitrogen and phosphorus removal effects. (2) The suitable growth temperature for microalgae is 25-28℃. When the ambient temperature is less than 4℃, microalgae growth almost stops. When the temperature is less than 15℃, microalgae activity is poor and growth is slow. Although microalgae that grow in or are found in Antarctica and the Arctic can grow in low-temperature environments, they are not very good at utilizing nutrients such as nitrogen and phosphorus in wastewater.

[0008] The low-temperature environment in cold regions severely affects the absorption of nitrogen and phosphorus by microalgae and their resource utilization. Therefore, this invention focuses on the development of low-temperature algal strains, seeking low-temperature tolerant microalgal strains with good tolerance to specific biogas slurries. These strains can grow in specific biogas slurries within a certain concentration range, absorbing ammonia nitrogen and phosphorus pollutants from the biogas slurry as their own nutrient sources, and exhibiting tolerance to toxic and harmful substances such as ammonia nitrogen, pathogenic microorganisms, and drug residues in aquaculture feed in the biogas slurry.

[0009] In a first aspect, the present invention provides a Spirulina, wherein the Spirulina W2 has the accession number CGMCC No. 40697. The Spirulina provided by the present invention was deposited on May 31, 2023, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 40697 and classification name Rhizomastix sp.

[0010] The optimal culture temperature for Spirulina provided by this invention is 5-25℃.

[0011] More specifically, the spirulina provided by the present invention is a low-temperature resistant spirulina, wherein the growth temperature of the low-temperature resistant spirulina is 5-15℃.

[0012] The Spirulina provided by this invention exhibits a doubling time of 2 days in biogas slurry at a low temperature of 15°C. -1 -2.4d -1 .

[0013] The spirulina provided by this invention is a high ammonia nitrogen-tolerant spirulina, which can survive in an environment with an ammonia nitrogen concentration higher than 400 mg / L.

[0014] Secondly, the present invention provides a wastewater treatment product containing a spirulina strain or spirulina fermentation broth; the spirulina W2 has the preservation number CGMCC No. 40697.

[0015] Thirdly, the present invention provides the application of the above-mentioned Spirulina or the above-mentioned wastewater treatment products in improving the ammonia nitrogen removal rate and / or total phosphorus removal rate of biogas slurry; the biogas slurry is the product of anaerobic fermentation of livestock and poultry wastewater.

[0016] In the application provided by this invention, the biogas slurry contains NH4 + -N concentration is 400-600 mg / L, COD is 1000-2000 mg / L, and TP is 15-35 mg / L.

[0017] In the applications provided by this invention, the ambient temperature is 5-15℃.

[0018] Fourthly, the present invention provides the application of the above-mentioned Spirulina or the above-mentioned wastewater treatment products in reducing eutrophication of water bodies.

[0019] If untreated biogas slurry is directly discharged into water bodies, it will cause eutrophication because the biogas slurry contains a large amount of ammonia nitrogen and phosphorus. Treating the biogas slurry with the spirulina provided by this invention before discharge can effectively prevent eutrophication.

[0020] The beneficial effects of this invention are as follows:

[0021] This invention successfully screened microalgae that can grow in low-temperature (5-15℃) environments with good growth effects, and can grow in environments with biogas slurry concentrations above 400 mg / L with good ammonia nitrogen and phosphorus removal effects; the microalgae is Spirulina, with accession number CGMCCNo.40697.

[0022] The spirulina provided by this invention has the ability to withstand low temperatures and high concentrations of ammonia nitrogen and phosphorus, and can provide a green and safe method for treating pig manure biogas slurry with high ammonia nitrogen and high phosphorus in cold regions. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is an electron microscope image of the Spirulina W2 strain culture of the present invention.

[0025] Figure 2 This is the growth and development tree of Spirulina W2 of the present invention.

[0026] Figure 3This is a comparative growth curve of Spirulina (CGMCC No. 40697) at different temperatures according to the present invention.

[0027] Figure 4 This is the growth curve of Spirulina (CGMCC No. 40697) of this invention cultured in biogas slurry at 15℃.

[0028] Figure 5 The results show the ammonia nitrogen removal rate of Spirulina (CGMCC No. 40697) from this invention, cultured in biogas slurry at 15℃.

[0029] Figure 6 The results show the total phosphorus removal rate of Spirulina (CGMCC No. 40697) cultured in biogas slurry at 15℃. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] Example 1: Algal strain isolation, purification, and culture

[0032] Soil samples were collected from the surface layer of soil near a farm in Qitaihe City, Heilongjiang Province, which had been contaminated by livestock and poultry wastewater. The sampling time was late April (the ambient temperature of the sample was 0-20℃). The moist active soil samples were placed in clean plastic bottles, sealed, and then sent to our laboratory by express delivery with low temperature circulation and stored at 4℃ for later use.

[0033] Approximately 0.5g of soil sample was placed in a 24-well tissue culture dish, and BG11 liquid medium (the specific composition of BG11 medium is shown in Table 1) was added. The dish was then placed in a light incubator with adjustable environmental parameters for algal enrichment culture. After 3-4 days of culture, the color of the medium clearly changed from colorless to light green. Algal samples were then isolated and purified using the streak plate method on BG11 solid agar (prepared by adding 1.5%-2% agar powder to sterilized BG11 liquid medium) until a single algal colony was formed on the plate. A sterile single algal colony was then picked and inoculated into liquid BG11 medium and cultured in a light incubator.

[0034] To ensure the purity of the algal strain, it was further purified using a 96-well plate serial dilution method. 100 μL of algal culture was placed in a 96-well plate, and 100 μL of sterile BG11 medium was added and mixed. 100 μL of the diluted algal culture was then used for serial dilution until a single algal cell grew from the last dilution. The algal culture from this well was then aspirated for expansion culture and the algal strain was fixed and preserved on a plate.

[0035] Use an optical microscope to observe whether the cultured microalgae strains have consistent cell morphology. If they are consistent, the separation is achieved. If they are inconsistent, repeat the 96-well plate gradient dilution separation method to separate and purify the algae strain again until the cell morphology is uniform.

[0036] The algal strains were cultured in 100mL Erlenmeyer flasks (effective culture volume 50mL) using BG11 liquid medium. The culture conditions were: temperature 15±0.5℃, light intensity 200μmol / m². 2 / s, light cycle 12:12, shake the bottle manually three times a day.

[0037] Example 2: Algal Species Identification

[0038] During the process of screening algae in cold soils, 16 algae were initially screened from different soil samples. After enrichment, separation and purification, only 6 dominant algae survived. One of them was identified as Spirulina and named W2.

[0039] Algal identification is performed in two steps: first, a preliminary morphological observation, followed by molecular biological identification. The isolated and purified microalgal strains were observed and photographed using an optical microscope (10x eyepiece × 40x objective lens) to observe the cell morphology, size, and structural characteristics of the strains. See the image below for morphological observation of the algal strains under the optical microscope. Figure 1 .

[0040] For diversity analysis of eukaryotic microorganisms, the attribution of samples can be determined at a higher level by analyzing the 18S rDNA sequence.

[0041] a) Extraction and PCR amplification of genomic DNA

[0042] Genomic DNA was extracted from the samples using CTAB or SDS methods, and the purity and concentration of the extracted DNA were then detected by agarose gel electrophoresis. Subsequently, an appropriate amount of DNA was placed in a centrifuge tube and diluted with sterile water to 1 ng / μl.

[0043] Using diluted genomic DNA as a template, specific primers with barcodes for the 18S V4 region (528F and 706R) were selected according to the amplification region. PCR was performed using Phusion High-Fidelity PCR Master Mix with GC Buffer from New England Biolabs and a high-efficiency, high-fidelity enzyme to ensure amplification efficiency and accuracy.

[0044] b) Mixing and purification of PCR products

[0045] PCR products were detected by 2% agarose gel electrophoresis. Equal volumes of PCR products were mixed according to their concentrations and thoroughly mixed before being detected again by 2% agarose gel electrophoresis. The target band was recovered using a gel recovery kit provided by Qiagen.

[0046] c) Library construction and sequencing

[0047] Library construction was performed using the NEBNext Ultra II DNA Library Prep Kit. The constructed library was then quantified using Qubit and Q-PCR. Once the library was deemed suitable, it was sequenced using a NovaSeq 6000.

[0048] The sequencing process was outsourced to Beijing Apsilon Biotechnology Co., Ltd. The obtained sequences were analyzed using BLAST in the GenBank database (http: / / www.ncbi.nlm.nih.gov / ) for homology detection, compared with existing algal gene sequences in the algal gene bank, and finally the species was determined. Its growth and development tree is shown below. Figure 2 As shown.

[0049] Example 3: Preservation of Algal Strains and Verification of Cold Resistance and Stress Tolerance

[0050] The Spirulina strain W2, which was isolated, purified, and identified in Examples 1 and 2, was deposited on May 31, 2023, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 40697 and classified as Rhizomastix sp.

[0051] To verify that the Spirulina strain CGMCC No.40697 isolated, purified, and preserved above has cold resistance and stress resistance and has potential in purifying pig manure biogas slurry, this invention has successively carried out temperature comparison experiments using pure culture medium and low-temperature experiments using real biogas slurry.

[0052] The culture medium used for the isolation and purification of algal strains was the standard BG11 medium, the formula of which is shown in Table 1.

[0053] Table 1. BG11 Culture Medium Formulation

[0054]

[0055] Note: After the culture medium is prepared, adjust the pH to 7.1 with 1 mol / L HCl or NaOH, and then autoclave it for later use.

[0056] The algal strain used in the experiment was the algal strain purified in Example 1 (accession number CGMCC No. 40697). In addition, two control algal strains were introduced. C-1 was *Stichococcus antarticus* (FACHB-2327), purchased from the Freshwater Algal Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). It is a low-temperature tolerant algal strain, screened from Fildes Peninsula, Antarctica, with original accession number UA4. C-2 was *Chlamydomonas* sp., a laboratory-existing algal strain with accession number CGMCC No. 15497. It is an algal strain that grows well at room temperature and has been disclosed in Chinese patent CN109251866B.

[0057] (1) Growth status of algae at different temperatures

[0058] Two sets of experiments were designed: one at a low temperature of 15℃ and the other at room temperature of 25℃, both conducted in a constant-temperature, light-controlled incubator. The reactors used were 250mL Erlenmeyer flasks with a working volume of 150mL. The culture conditions were anaerobic under light, using a sequencing batch culture method. Initial OD of inoculation was... 680 The value is 0.40±0.01, and the light intensity is 200 μmol / m². 2 / s., with a light cycle (L:D) of 12:12, and cultured until the stationary phase and then until the death phase.

[0059] Comparison of the growth of three microalgae in BG11 medium at different temperatures: Figure 3 As shown. During the 12-day culture period, all three microalgae were able to grow to varying degrees in BG11 medium at different temperatures. Among them, Spirulina strain CGMCC No.40697 showed good growth under both normal temperature (25℃) and low temperature (15℃) conditions.

[0060] Under low temperature (15℃) conditions, the biomass of Spirulina strain CGMCC No.40697 reached 273.54 mg / L on day 12; followed by Spica fusiforme strain FACHB-2327, whose biomass on day 12 was 253.22 mg / L; and Chlamydomonas strain CGMCC No.15497 showed the worst growth, with a biomass of 227.63 mg / L on day 12.

[0061] The experimental results show that, compared with the low-temperature pyrophylloides strain purchased from commercial algal strain banks and the Chlamydomonas strain previously preserved by our research group, the Spirulina strain isolated and purified by this invention has cold resistance and stress resistance, has growth potential under low temperature conditions, and has the strongest ability to accumulate biomass.

[0062] (2) The isolated and purified algae strains were used to purify pig manure biogas slurry.

[0063] The anaerobic fermentation slurry from pig manure was taken from a biogas digester in Harbin, Heilongjiang Province. The main raw material for the anaerobic fermentation in this biogas digester was manure from pig farms in surrounding villages. Before inoculating the slurry with microalgae, the slurry was centrifuged at 10,000 rpm for 10 minutes. The supernatant was then filtered through a 0.45 μm membrane to further remove impurities. After high-temperature and high-pressure sterilization, the treated slurry was stored in a 4°C refrigerator away from light for later use. Because the ammonia nitrogen and other related indicators in the pig manure slurry were relatively high, which would inhibit the growth of microalgae, the slurry was diluted 10 times and used as experimental water. The water quality characteristics after 10-fold dilution are detailed in Table 2.

[0064] Table 2. Water quality characteristics of biogas slurry after 10-fold dilution

[0065]

[0066]

[0067] The algal strain used in the experiment was the one isolated and purified in Example 1 (preservation number CGMCC No. 40697), which was cultured in BG11 medium to the logarithmic growth phase before being used in this study. The biogas slurry used in the experiment was anaerobic fermentation biogas slurry of pig manure diluted 10 times, and its properties are shown in Table 2.

[0068] The reactor used in the experiment was a 250mL Erlenmeyer flask, with a working volume of 150mL. The culture conditions were anaerobic under light, and the culture method was sequencing batch culture. Initial OD after inoculation... 680 The value was 0.40±0.01, the culture temperature was 15℃, and the light intensity was 200μmol / m². 2 / s, the photoperiod (L:D) was 12:12, the culture time was 12 days, and the bottles were manually shaken three times a day.

[0069] The test results are as follows:

[0070] At 15℃, the growth of three microalgae in pig manure biogas slurry was compared as follows: Figure 4 As shown in the figure, all three microalgae were able to grow to varying degrees in pig manure biogas slurry during the 12-day cultivation period. Among them, Spirulina strain CGMCC No.40697 showed the best growth effect in pig manure biogas slurry, with a biomass of 207.30 mg / L on the 12th day; the biomass of Hypothermophyta strain FACHB-2327 was 191.12 mg / L on the 12th day; and the growth of Chlamydomonas strain CGMCC No.15497 was the worst, with a biomass of 143.63 mg / L on the 12th day.

[0071] At a low temperature of 15℃, the doubling time of Spirulina strain CGMCC No. 40697 in biogas slurry was 2.37 days. -1 .

[0072] The experimental results show that, compared with the low-temperature pyrophylloides strains randomly selected from commercial algal strain banks and the Chlamydomonas strains previously preserved by our research group, the Spirulina strain isolated and purified by this invention has the best tolerance in pig manure biogas slurry and the strongest ability to accumulate biomass.

[0073] At 15℃, three microalgae strains were used to treat pig manure biogas slurry. The changes in ammonia nitrogen in the pig manure biogas slurry were as follows: Figure 5 As shown, during the 12-day cultivation period, all three microalgae strains were able to remove ammonia nitrogen from pig manure biogas slurry to varying degrees. Among them, Spirulina strain CGMCC No. 40697 showed the strongest removal capacity for ammonia nitrogen from pig manure biogas slurry, with a removal rate of 44.93% after 12 days; followed by Chilodonella strain FACHB-2327, with a removal rate of 43.67% after 12 days; and Chlamydomonas strain CGMCC No. 15497 showed the weakest removal capacity for ammonia nitrogen, with a removal rate of only 42.94% after 12 days.

[0074] At 15℃, three microalgae strains were used to treat pig manure biogas slurry. The changes in total phosphorus in the pig manure biogas slurry were as follows: Figure 6 As shown, all three microalgae strains were able to absorb and remove total phosphorus from pig manure biogas slurry to varying degrees, and most of the phosphorus was degraded by around day 4, after which the TP concentration gradually stabilized.

[0075] During the 12-day cultivation period, Spirulina strain CGMCC No.40697 achieved a total phosphorus removal rate of 8.97% in pig manure biogas slurry, while the low-temperature Spica strain FACHB-2327 achieved a total phosphorus removal rate of 5.03%. Chlamydomonas strain CGMCC No.15497 showed the worst removal capacity for total phosphorus in pig manure biogas slurry, with a removal rate of only 3.15%.

[0076] In summary, the Spirulina strain CGMCC No. 40697 isolated and purified by this invention exhibits good tolerance to certain concentrations of pig manure biogas slurry. Cultivating this microalgae in pig manure biogas slurry can effectively purify the slurry, demonstrating significant denitrification and phosphorus removal. Under low-temperature (15℃) conditions, compared with low-temperature-sensitive *Spirometra* strains randomly selected from commercial algal banks and *Chlamydomonas* strains previously preserved by our research group, this wild-type strain isolated from the natural environment exhibits better growth capacity in pig manure biogas slurry. Therefore, it is an ideal microbial material for treating pig manure biogas slurry in cold regions, solving the problem of inhibited microbial growth in low-temperature environments and possessing broad application prospects.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Spirulina ( Rhizomastix sp.)W2, characterized in that, The accession number of the Spirulina W2 is CGMCC No. 40697.

2. A wastewater treatment product, characterized in that, The strain or liquid containing Spirulina W2; the preservation number of Spirulina W2 is CGMCC No. 40697; The wastewater is pig manure biogas slurry.

3. The application of the Spirulina W2 of claim 1 or the wastewater treatment product of claim 2 in improving the ammonia nitrogen removal rate and / or total phosphorus removal rate of biogas slurry; wherein the biogas slurry is pig manure biogas slurry.

4. Use according to claim 3, characterized in that, The biogas slurry contains NH4 + -N concentration is 400-600 mg / L, COD is 1000-2000 mg / L, and TP is 15-35 mg / L.

5. Use according to claim 3 or 4, characterized in that, The ambient temperature for this application is 15±0.5℃.

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