Chlorella sorokiniana and its application in livestock and poultry wastewater treatment in cold regions

Abstract

The present application relates to the technical field of wastewater treatment, and particularly provides a low-temperature-resistant chlamydomonas reinhardtii and application thereof in livestock and poultry wastewater treatment in cold regions. Through development of low-temperature algae, the present application obtains a chlamydomonas reinhardtii strain which can grow in an environment with a biogas slurry concentration higher than 400 mg / L under low-temperature conditions and has good ammonia nitrogen and phosphorus removal effects. The chlamydomonas reinhardtii provided by the present application has a preservation number of CGMCC No.40692, a preservation number of CGMCC No.40693, a preservation number of CGMCC No.40694 or a preservation number of CGMCC No.40695. The chlamydomonas reinhardtii provided by the present application is a cold-resistant and stress-resistant algae strain for treating high-ammonia-nitrogen and high-phosphorus pig manure biogas slurry in cold regions and cold-like regions, and can reduce the pressure of livestock and poultry wastewater treatment and resourceization in high-cold regions.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment technology, and in particular to Chlamydomonas aeruginosa, a cold-resistant algae, and its application in the treatment of livestock and poultry wastewater in cold regions. Background Technology

[0002] In recent years, with the rapid development of large-scale and intensive livestock farming, the pollution problem of livestock manure has become increasingly serious. The Food and Agriculture Organization of the United Nations has listed intensive livestock farming as one of the three major sources of pollution in the world's environment. Pig farm wastewater is considered one of the most polluting agricultural and industrial wastewaters. It contains high concentrations of organic matter, volatile fatty acids (carbon sources), ammonia nitrogen (nitrogen sources), and even heavy metals (such as copper, lead, and zinc). If left untreated or improperly treated, pig farm wastewater discharge poses a potential threat to environmental safety, leading to foul odors, eutrophication of nearby water sources, and increased carbon emissions. Developing an efficient method for treating pig farm wastewater is a global concern.

[0003] Large and medium-sized biogas projects using livestock manure as raw material are the most common treatment method. Utilizing biogas engineering technology to treat livestock waste can not only control pollution but also realize the resource utilization of waste, achieving triple benefits in energy, environmental protection, and economy, which is in line with the concept of sustainable development of human society. However, the treatment of biogas slurry in large and medium-sized biogas projects still faces serious environmental problems. Biogas slurry that has only undergone anaerobic treatment still contains a large amount of nutrients such as nitrogen and phosphorus. Direct discharge into water bodies can easily cause eutrophication and secondary pollution, while also placing great pressure on local disposal.

[0004] Microalgae are autotrophic organisms that can absorb CO2 from the air under light and simultaneously synthesize their own algal cells using nutrients such as nitrogen and phosphorus from wastewater. They have a strong nitrogen and phosphorus conversion capacity and contain high-value substances such as oils and proteins, which can be used for energy production, as feed, and as fertilizer in agriculture, making them highly valuable resources. Artificial cultivation of microalgae requires a liquid culture medium containing sufficient carbon, nitrogen, and phosphorus, and the nutrient composition of some organic wastewater is similar to that of microalgae culture media.

[0005] According to literature, the cost of microalgae culture medium accounts for 30%-60% of the total cost of microalgae cultivation. Cultivating microalgae in wastewater allows the algae to absorb nutrients such as carbon, nitrogen, and phosphorus from the wastewater to promote their growth, thus purifying the wastewater in the process. The harvested algal cell biomass can then be used to produce biofuel, feed additives, or other high-value-added products, representing a green and low-cost industrial development approach for microalgae cultivation. Therefore, using microalgae to treat wastewater can remove pollutants, fix carbon, and produce high-value substances simultaneously, making it a sustainable wastewater treatment technology with broad development prospects.

[0006] Among alternatives to pig manure biogas slurry treatment, microalgae-driven bioprocesses are promising due to their cost-effectiveness and high energy efficiency potential. Integrating microalgae systems into pig manure biogas slurry treatment processes can purify wastewater and recover resources, making it a carbon-friendly process that can simultaneously reduce carbon emissions, recover resources, and protect the environment.

[0007] Biogas slurry from cold-region biogas projects is rich in nutrients such as nitrogen and phosphorus, and these nutrients need to be recovered. 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, which seriously restricts the efficiency of livestock and poultry wastewater treatment and resource utilization. Therefore, how to carry out low-cost and in-depth treatment of livestock and poultry wastewater on-site, especially the research on the resource utilization of carbon, nitrogen, and phosphorus under the low-temperature effect, is one of the main problems to be solved in the resource utilization treatment of livestock and poultry wastewater in cold regions.

[0008] There are many mechanistic and technical problems in using microalgae to treat livestock and poultry wastewater in cold regions: (1) High concentrations of ammonia nitrogen in livestock and poultry wastewater inhibit the growth of microalgae; (2) The suitable growth temperature for microalgae is 25-28℃. When the ambient temperature is less than 4℃, microalgae growth almost stops, and when the ambient temperature is less than 15℃, microalgae growth is slow. The low temperature environment in cold regions seriously affects the absorption of nitrogen and phosphorus by microalgae and their resource utilization. In the existing technology, there are no low-temperature algae species (5-15℃) that can grow in an environment where the ammonia nitrogen concentration in the biogas slurry is higher than 400 mg / L and have good ammonia nitrogen and phosphorus removal effects.

[0009] To address the aforementioned issues, it is necessary to develop low-temperature algal strains and identify microalgae strains with good tolerance to specific biogas slurries. These strains should be able to grow in specific biogas slurries within a certain concentration range, consuming nitrogen and phosphorus pollutants from the biogas slurry as their own nutrient source, and exhibiting tolerance to toxic and harmful substances such as ammonia nitrogen, pathogenic microorganisms, and drug residues in aquaculture feed. Summary of the Invention

[0010] This invention provides a low-temperature resistant Chlamydomonas and its application in the treatment of livestock and poultry wastewater in cold regions.

[0011] In a first aspect, the present invention provides Chlamydomonas, wherein the preservation number of Chlamydomonas is CGMCC No. 40692, or the preservation number of Chlamydomonas is CGMCC No. 40693, or the preservation number of Chlamydomonas is CGMCC No. 40694, or the preservation number of Chlamydomonas is CGMCC No. 40695.

[0012] More specifically, the Chlamydomonas W3 obtained by screening in this 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. 40692 and classification name: Chlamydomonas sp.

[0013] The Chlamydomonas W4 obtained by screening in this 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. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. The deposit number is CGMCC No. 40693, and the classification name is Chlamydomonas sp.

[0014] The Chlamydomonas W5 strain obtained by this 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. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The deposit number is CGMCC No. 40694, and the classification name is Chlamydomonas sp.

[0015] The Chlamydomonas W6 obtained by screening in this 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. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The deposit number is CGMCC No. 40695, and the classification name is Chlamydomonas sp.

[0016] The Chlamydomonas provided by this invention grows well at a culture temperature of 5-25℃.

[0017] More specifically, the Chlamydomonas provided by this invention is a low-temperature resistant Chlamydomonas, and the growth temperature of the low-temperature resistant Chlamydomonas is 5-15℃. The biomass of the Chlamydomonas provided by this invention can reach 261.19-275.12 mg / L on the 12th day under low temperature (15℃) conditions.

[0018] The *Chlamydomonas* species provided by this invention exhibits a doubling time of 1.91 days in biogas slurry under low-temperature (15°C) conditions. -1 -2.23d -1 .

[0019] Secondly, the present invention provides a wastewater treatment product containing Chlamydomonas strain or Chlamydomonas liquid; wherein the Chlamydomonas is one or more of the following Chlamydomonas species with accession numbers CGMCC No. 40692, CGMCC No. 40693, CGMCC No. 40694 and CGMCC No. 40695.

[0020] Thirdly, the present invention provides the application of the above-mentioned Chlamydomonas or the above-mentioned wastewater treatment products in improving the removal of ammonia nitrogen and / or total phosphorus from wastewater.

[0021] In the application provided by this invention, the wastewater is biogas slurry, and the biogas slurry contains NH4. + -N concentration is higher than 400 mg / L.

[0022] More specifically, the wastewater provided by this invention has a COD of 1000-2000 mg / L and NH4+. + -N is 400-600 mg / L, and TP is 15-35 mg / L.

[0023] In the application provided by this invention, the ambient temperature is between 5-15°C.

[0024] This invention utilizes high-ammonia-nitrogen and high-phosphorus pig manure biogas slurry from cold regions to screen for cold-resistant and stress-tolerant algal species, thereby obtaining information on the NH4+ content in the biogas slurry under low-temperature conditions. + Chlamydomonas strain that grows in environments with -N concentrations above 400 mg / L and exhibits good ammonia and phosphorus removal effects.

[0025] Fourthly, the present invention also provides the application of the above-mentioned Chlamydomonas or the above-mentioned wastewater treatment products in reducing eutrophication of water bodies.

[0026] Fifthly, the present invention provides a wastewater treatment method, which uses the above-mentioned Chlamydomonas or the above-mentioned wastewater treatment product to treat wastewater; preferably, the wastewater is biogas slurry from the anaerobic fermentation of pig manure.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention, through field sampling (soil screening) and low-temperature screening, has shown that it can grow in a low-temperature, high-concentration biogas slurry environment and exhibits good growth performance (doubling time of 1.91 days). -1 -2.23d -1 The selected Chlamydomonas species, with accession numbers CGMCC No. 40692, CGMCC No. 40693, CGMCC No. 40694, or CGMCC No. 40695, can grow in environments with biogas slurry concentrations higher than 400 mg / L and exhibit good ammonia nitrogen and phosphorus removal effects.

[0029] The Chlamydomonas provided by this invention can treat pig manure biogas slurry with high ammonia nitrogen and high phosphorus in cold regions, thereby reducing the pressure on livestock and poultry wastewater treatment and resource utilization in high-altitude and cold regions. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in this invention or the prior art, the appendices used in the description of the embodiments or the prior art will be explained below. Figure 1 As will be briefly introduced, it is obvious that the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort.

[0031] Figure 1 This is an electron microscope image of the Chlamydomonas W3 strain cultured according to the present invention.

[0032] Figure 2 This is an electron microscope image of the Chlamydomonas W4 strain cultured according to the present invention.

[0033] Figure 3 This is an electron microscope image of the Chlamydomonas W5 strain cultured according to the present invention.

[0034] Figure 4 This is an electron microscope image of the Chlamydomonas W6 strain cultured according to the present invention.

[0035] Figure 5 This is the growth and development tree of Chlamydomonas W3 in this invention.

[0036] Figure 6 This is the growth and development tree of Chlamydomonas W4 in this invention.

[0037] Figure 7 This is the growth and development tree of Chlamydomonas W5 in this invention.

[0038] Figure 8 This is the growth and development tree of Chlamydomonas W6 in this invention.

[0039] Figure 9 This is a comparison of the growth curves of Chlamydomonas W3 (CGMCC No. 40692) at different temperatures.

[0040] Figure 10 This is a comparison of the growth curves of Chlamydomonas W4 (CGMCC No. 40693) at different temperatures.

[0041] Figure 11 This is a comparison of the growth curves of Chlamydomonas W5 (CGMCC No. 40694) at different temperatures.

[0042] Figure 12This is a comparison of the growth curves of Chlamydomonas W6 (CGMCC No. 40695) at different temperatures.

[0043] Figure 13 This is the growth curve of Chlamydomonas W3 (CGMCC No.40692) of this invention in a biogas slurry culture comparative experiment at 15℃.

[0044] Figure 14 This is the growth curve of Chlamydomonas W4 (CGMCC No. 40693) of this invention in a biogas slurry culture comparative experiment at 15℃.

[0045] Figure 15 This is the growth curve of Chlamydomonas W5 (CGMCC No.40694) of this invention in a biogas slurry culture comparative experiment at 15℃.

[0046] Figure 16 The growth curve of Chlamydomonas W6 (CGMCC No. 40695) of this invention in biogas slurry culture comparison test at 15℃ is shown.

[0047] Figure 17 The ammonia nitrogen removal effect of Chlamydomonas W3 (CGMCC No.40692) in biogas slurry culture was compared at 15℃.

[0048] Figure 18 The ammonia nitrogen removal effect of Chlamydomonas W4 (CGMCC No.40693) in biogas slurry culture was compared at 15℃.

[0049] Figure 19 The ammonia nitrogen removal effect of the Chlamydomonas W5 (CGMCC No.40694) cultured in biogas slurry at 15℃ is compared with that of the present invention.

[0050] Figure 20 The ammonia nitrogen removal effect of Chlamydomonas W6 (CGMCC No.40695) in biogas slurry culture was compared at 15℃.

[0051] Figure 21 The total phosphorus removal effect of Chlamydomonas W3 (CGMCC No.40692) in biogas slurry culture was compared at 15℃.

[0052] Figure 22 The total phosphorus removal effect of Chlamydomonas W4 (CGMCC No. 40693) in biogas slurry culture was compared at 15℃.

[0053] Figure 23 The total phosphorus removal effect of the Chlamydomonas W5 (CGMCC No.40694) cultured in biogas slurry at 15℃ is compared with that of the present invention.

[0054] Figure 24The total phosphorus removal effect of the Chlamydomonas W6 (CGMCC No.40695) cultured in biogas slurry at 15℃ is compared with that of the present invention. Detailed Implementation

[0055] 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.

[0056] Example 1: Sample Collection and Algal Strains Isolation

[0057] (1) Sample collection

[0058] The soil samples used in this invention were soil samples from a village near a biogas digester in Harbin, Heilongjiang Province, which had been contaminated by anaerobic fermentation of pig manure and biogas slurry, and the surface soil samples from a farm near Qitaihe, Heilongjiang Province, which had been contaminated by livestock and poultry wastewater. The sampling time was late April (the ambient temperature of the samples was 0-20℃). The moist active soil samples were placed in clean plastic bottles, sealed, and then sent to the laboratory by low-temperature circulating express delivery and stored at 4℃ for later use.

[0059] (2) Algal strain isolation, purification and culture

[0060] 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.

[0061] 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.

[0062] Use an optical microscope to observe whether the cell morphology of the cultured microalgae strains is consistent. If they are consistent, the purpose of separation has been achieved. If they are inconsistent, repeat the above work until the cell morphology is uniform.

[0063] The culture containers used during the algal strain cultivation stage were 100mL Erlenmeyer flasks (effective culture volume of 50mL), BG11 liquid medium, and the culture conditions were: temperature 15±0.5℃, light intensity 200μmol / m2 / s, photoperiod 12:12, and manual shaking of the flasks three times a day.

[0064] Table 1. BG11 Culture Medium Formulation

[0065]

[0066] 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.

[0067] Example 2: Algal Species Identification and Preservation

[0068] After screening and cultivation as described in Example 1, algal strains that could grow stably under low-temperature conditions were identified. During the algal screening process in cold-region soils, 16 algal strains were initially screened from different soil samples. After enrichment, separation, and purification, only 6 dominant algal strains survived, and 4 of them were identified as Chlamydomonas.

[0069] The identification of algal strains was carried out in two steps: first, preliminary morphological observation, and then 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, structure, and other characteristics of the algal strains. Morphological images of algal strains W3, W4, W5, and W6 isolated in Example 1 are shown below. Figure 1 , Figure 2 , Figure 3 , Figure 4 .

[0070] For diversity analysis of eukaryotic microorganisms, the attribution of samples can be determined at a higher level by analyzing the 18S rDNA sequence.

[0071] (1) Extraction of genomic DNA and PCR amplification

[0072] 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.

[0073] 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.

[0074] a) Mixing and purification of PCR products

[0075] 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.

[0076] b) Library construction and sequencing

[0077] 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 NovaSeq6000.

[0078] 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, and compared with existing algal gene sequences in the algal gene bank to determine the species. The growth and development tree of the isolated algal strain in Example 1 is shown below. Figure 5 , Figure 6 , Figure 7 , Figure 8 .

[0079] (2) Preservation of algal strains

[0080] The Chlamydomonas strains W3, W4, W5, and W6, which were isolated, purified, and identified in Examples 1 and 2, were deposited on May 31, 2023, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession numbers CGMCC No. 40692, CGMCC No. 40693, CGMCC No. 40694, and CGMCC No. 40695.

[0081] To verify that the isolated, purified, and preserved algal strains CGMCC No. 40692, CGMCC No. 40693, CGMCC No. 40694, and CGMCC No. 40695 possess cold resistance and stress tolerance and have 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.

[0082] Example 3: Verification of the cold resistance and stress tolerance of isolated and purified algal strains

[0083] The culture medium used in the experiment was standard BG11 medium, the formula of which is shown in Table 1. The algal strains used in the experiment were Chlamydomonas sp. W3, W4, W5, and W6 (accession numbers CGMCC No. 40692, CGMCC No. 40693, CGMCC No. 40694, and CGMCC No. 40695) purified in Example 1. In addition, two control algal strains were introduced. C-1 was Stichococcus antarticus (FACHB-2327), purchased from the Freshwater Algae 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 the original accession number UA4. C-2 was Chlamydomonas sp., an existing laboratory species, with the accession number CGMCC No. 15497. It is an algal strain that grows well at room temperature and has been disclosed in Chinese Patent CN109251866B.

[0084] (1) Growth status of algae at different temperatures

[0085] Two sets of experiments were designed: one set at a low temperature of 15℃ and the other set at a normal temperature of 25℃, both conducted in a constant temperature and light incubator.

[0086] 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 concentration was 0.40±0.01, the light intensity was 200 μmol / m2 / s, and the photoperiod (L:D) was 12:12. The cells were cultured until the stationary phase and then until the death phase.

[0087] (2) Separation and purification of algae strains to purify pig manure biogas slurry

[0088] 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.

[0089] Table 2. Water quality characteristics of biogas slurry after 10-fold dilution

[0090]

[0091] The algal strains used in this experiment were those purified in Example 1 (CGMCC No. 40692, CGMCC No. 40693, CGMCC No. 40694, and CGMCC No. 40695), and were 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 slurry of pig manure diluted 10 times, and its properties are shown in Table 2.

[0092] 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 concentration was 0.40±0.01, the culture temperature was 15℃, the light intensity was 200μmol / m2 / s, the photoperiod (L:D) was 12:12, the culture time was 12d, and the flask was manually shaken three times a day.

[0093] The test results are as follows:

[0094] Comparison of microalgae growth in BG11 medium at different temperatures: Figure 9 , Figure 10 , Figure 11 , Figure 12 As shown, during the 12-day culture period, microalgae were able to grow to varying degrees in BG11 medium at different temperatures.

[0095] Among them, Chlamydomonas strain CGMCC No.40692 showed good growth under both normal temperature (25℃) and low temperature (15℃) conditions. Under low temperature (15℃) conditions, the biomass reached 261.19 mg / L on the 12th day.

[0096] Chlamydomonas strain CGMCC No.40693 showed good growth under both normal temperature (25℃) and low temperature (15℃) conditions. Under low temperature (15℃) conditions, the biomass reached 275.12 mg / L on the 12th day.

[0097] Chlamydomonas strain CGMCC No.40694 showed good growth under both normal temperature (25℃) and low temperature (15℃) conditions. Under low temperature (15℃) conditions, the biomass reached 272.18 mg / L on the 12th day.

[0098] Chlamydomonas strain CGMCC No.40695 showed good growth under both normal temperature (25℃) and low temperature (15℃) conditions. Under low temperature (15℃) conditions, the biomass reached 267.59 mg / L on the 12th day.

[0099] The biomass of the low-temperature pyrophylloides strain FACHB-2327 was 253.22 mg / L on day 12 under low-temperature (15℃) conditions. The Chlamydomonas strain CGMCC NO.15497 showed the worst growth under low-temperature conditions, with a biomass of 227.63 mg / L on day 12 under low-temperature (15℃) conditions.

[0100] 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 Chlamydomonas strains isolated and purified by this invention have cold resistance and stress resistance, have growth potential under low-temperature conditions, and have the strongest ability to accumulate biomass.

[0101] The growth status of microalgae in pig manure biogas slurry at a low temperature of 15℃ is as follows: Figure 13 , Figure 14 , Figure 15 , Figure 16 As shown, the microalgae were able to grow to varying degrees in the pig manure biogas slurry during the 12-day cultivation period. Among them, Chlamydomonas strain CGMCC No. 40692 achieved a biomass of 200.38 mg / L in the pig manure biogas slurry on day 12; Chlamydomonas strain CGMCC No. 40693 showed the best growth effect, with a biomass of 223.79 mg / L on day 12; Chlamydomonas strain CGMCC No. 40694 achieved a biomass of 210.77 mg / L on day 12; and Chlamydomonas strain CGMCC No. 40695 achieved a biomass of 206.02 mg / L on day 12.

[0102] The biomass of the low-temperature pyrophylloides strain FACHB-2327 in pig manure biogas slurry was 191.12 mg / L on day 12, while the growth of the Chlamydomonas strain CGMCC NO.15497 was the worst, with a biomass of 143.63 mg / L on day 12.

[0103] At 15℃, the doubling time of Chlamydomonas strains CGMCC No. 40692, CGMCC No. 40693, CGMCC No. 40694, and CGMCC No. 40695 in biogas slurry was 2.23 days. -1 1.91d -1 2.12d -1 2.23d -1 .

[0104] The experimental results show that, compared with the low-temperature *Schizophyllum commune* strains randomly selected from commercial algal strain banks and the *Chlamydomonas* strains previously preserved by our research group, the *Chlamydomonas* strains isolated and purified in this invention exhibit the best tolerance in pig manure biogas slurry and the strongest ability to accumulate biomass.

[0105] At 15℃, microalgae were used to treat pig manure biogas slurry, and the changes in ammonia nitrogen in the slurry were as follows: Figure 17 , Figure 18 , Figure 19 , Figure 20 As shown, at a low temperature of 15℃, during a 12-day cultivation period, Chlamydomonas W3-W6 were able to remove ammonia nitrogen from pig manure biogas slurry to varying degrees.

[0106] Among them, Chlamydomonas strain CGMCC No.40692 achieved a 46.49% ammonia nitrogen removal rate in pig manure biogas slurry after 12 days.

[0107] Chlamydomonas strain CGMCC No.40693 showed the strongest removal capacity for ammonia nitrogen in pig manure biogas slurry, with a removal rate of 47.02% after 12 days.

[0108] Chlamydomonas strain CGMCC No. 40694 achieved a 44.93% ammonia nitrogen removal rate in pig manure biogas slurry after 12 days.

[0109] Chlamydomonas strain CGMCC No. 40695 achieved a 44.09% ammonia nitrogen removal rate in pig manure biogas slurry after 12 days.

[0110] The low-temperature pyrophylloides strain FACHB-2327 achieved a 43.67% removal rate of ammonia nitrogen from pig manure biogas slurry after 12 days, while the Chlamydomonas strain CGMCC NO.15497 showed the worst ammonia nitrogen removal capacity, with a removal rate of only 42.94% after 12 days.

[0111] At 15℃, microalgae were used to treat pig manure biogas slurry, and the changes in total phosphorus in the pig manure biogas slurry were as follows: Figure 21 , Figure 22 , Figure 23 , Figure 24As shown, at a low temperature of 15℃, Chlamydomonas W3-W6 were able to remove ammonia nitrogen from pig manure biogas slurry to varying degrees, and most of the phosphorus was basically degraded around the 4th day, after which the TP concentration gradually remained stable.

[0112] During the 12-day cultivation period, the removal rate of total phosphorus in pig manure biogas slurry was as follows: Chlamydomonas strain CGMCC No. 40692 was 9.52%, Chlamydomonas strain CGMCC No. 40693 was 15.64%, Chlamydomonas strain CGMCC No. 40694 was 7.24%, Chlamydomonas strain CGMCC No. 40695 was 9.88%, the removal rate of total phosphorus in pig manure biogas slurry was 5.03%, and Chlamydomonas strain CGMCC No. 15497 had the worst removal capacity, with a total phosphorus removal rate of only 3.15%.

[0113] In summary, the *Chlamydomonas* strains (CGMCC No. 40692, CGMCC No. 40693, CGMCC No. 40694, and CGMCC No. 40695) isolated and purified by this invention exhibit good tolerance to certain concentrations of pig manure biogas slurry. Cultivating these microalgae in pig manure biogas slurry can effectively purify the slurry, demonstrating significant denitrification and phosphorus removal. Under low-temperature (15℃) conditions, compared to *Spirometra* strains purchased from commercial algal banks and *Chlamydomonas* strains previously preserved by our research group, the wild-type algae strains isolated from the natural environment involved in this invention show better growth capacity in pig manure biogas slurry. Therefore, they are 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.

[0114] 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. Chlamydomonas ( chlamydomonas sp. ), characterized in that, The accession number of the *Chlamydomonas* is CGMCC No. 40692, or CGMCC No. 40693, or CGMCC No. 40694, or CGMCC No. 40695.

2. A wastewater treatment product, characterized in that, Contains Chlamydomonas strains or Chlamydomonas liquid; the Chlamydomonas is one or more of the Chlamydomonas species with accession numbers CGMCC No. 40692, CGMCC No. 40693, CGMCC No. 40694 and CGMCC No. 40695.

3. The application of the Chlamydomonas as described in claim 1 or the wastewater treatment product as described in claim 2 in improving the removal of ammonia nitrogen and / or total phosphorus from wastewater.

4. The application according to claim 3, characterized in that, The wastewater is biogas slurry, and the biogas slurry contains NH4. + -N concentration is higher than 400 mg / L.

5. The application according to any one of claims 3-4, characterized in that, The ambient temperature for this application is 15-25℃.

6. The application of Chlamydomonas as described in claim 1 or the wastewater treatment product as described in claim 2 in reducing eutrophication of water bodies.

7. A wastewater treatment method, characterized in that, Wastewater is treated using the Chlamydomonas as described in claim 1 or the wastewater treatment product as described in claim 2.

8. The wastewater treatment method according to claim 7, characterized in that, The wastewater is biogas slurry produced by the anaerobic fermentation of pig manure.

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