A strain of low-temperature-resistant micractinium and application thereof

Abstract

The present application relates to the technical field of livestock and poultry wastewater treatment, and specifically provides a low-temperature-resistant microchaete aeruginosum and application thereof.The present application screens the microchaete aeruginosum which is low-temperature-resistant and has high nitrogen and phosphorus concentration tolerance, and the preservation number is CGMCC No.40696.The microchaete aeruginosum with the preservation number CGMCC No.40696 provided by the present application can realize the microbial treatment of high-ammonia-nitrogen and high-phosphorus pig manure slurry in cold regions, reduce the pressure of livestock and poultry wastewater treatment and resourceization in high-cold regions, and reduce the degree of water eutrophication.

Description

Technical Field

[0001] This invention relates to the field of livestock and poultry wastewater treatment technology, specifically providing a low-temperature resistant strain of Mycorrhiza and its applications. Background Technology

[0002] Microalgae can absorb CO2 from the air under light and simultaneously synthesize their own algae using nutrients such as nitrogen and phosphorus in wastewater. In this process, they play a role in purifying wastewater. The harvested algal cell biomass can then be used as microalgae biofuel, feed additives, or other high-value-added products. This represents a green and low-cost approach to the industrialization of microalgae cultivation.

[0003] Pig farm wastewater is considered one of the most polluting agricultural and industrial wastewaters, especially in pig farms located in cold regions. The low temperatures in these areas lead to problems such as poor microbial activity, slow reactions, and low material conversion efficiency during treatment, severely restricting the efficiency of livestock wastewater treatment and resource recovery. Therefore, how to conduct low-cost, in-situ, intensive treatment of livestock wastewater, particularly research on the resource recovery of carbon, nitrogen, and phosphorus under low-temperature effects, is one of the main problems that urgently need to be solved in the resource recovery treatment of livestock wastewater in cold regions.

[0004] Using 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. However, the optimal growth temperature for common microalgae is 25-28℃. When the ambient temperature is below 4℃, microalgae growth almost stops. 4℃ is the critical temperature for the growth of most microorganisms, and microbial activity is poor and growth is slow when the growth temperature is below 15℃.

[0005] Microalgae that grow in or are found in the Antarctic and Arctic regions, while capable of growing in low-temperature environments, are not well able to utilize nutrients such as nitrogen and phosphorus in wastewater. Currently, there are no low-temperature algal species (5-15℃) that can grow in biogas slurry environments with ammonia nitrogen concentrations higher than 400 mg / L and exhibit good ammonia and phosphorus removal efficiency. Summary of the Invention

[0006] This invention provides a low-temperature resistant algae and its application to solve the difficulties existing in the treatment of sewage in cold regions, and to realize the microbial treatment of sewage in cold regions.

[0007] In a first aspect, the present invention provides a strain of Mycorrhizal maculatus, the preservation number of which is CGMCC No.40696.

[0008] The *Mycorrhizae* W1 strain obtained in this invention was deposited on May 31, 2023, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 40696, and it is classified as *Mycorrhizae*. Mychonastes sp.

[0009] The culture temperature of Mycorrhiza provided by this invention is 5-25℃.

[0010] The *McClella* provided by this invention is a low-temperature resistant *McClella*, and the growth temperature of the low-temperature resistant *McClella* is 5-15℃.

[0011] The doubling time of the *Mexicoplankton* provided by this invention in biogas slurry at 15°C is 2-2.2 days.

[0012] Secondly, the present invention provides a wastewater treatment product containing *McClella esculenta* strain or *McClella esculenta* fermentation broth; the preservation number of *McClella esculenta* is CGMCC No. 40696.

[0013] Thirdly, the present invention provides the application of the above-mentioned Myco-Algae or the above-mentioned wastewater treatment products in improving the removal of ammonia nitrogen and / or total phosphorus from wastewater.

[0014] In the application provided by this invention, the wastewater is pig manure wastewater, and the pig manure wastewater contains NH4 + -N concentration is 400-600 mg / L.

[0015] More specifically, the pig manure 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.

[0016] The ambient temperature for the application provided by this invention is 5-15℃.

[0017] Fourthly, the present invention provides the application of the above-mentioned Mycobacterium or the above-mentioned wastewater treatment products in reducing eutrophication of water bodies.

[0018] This invention provides a microalgae-driven pig manure wastewater treatment process, which integrates the microalgae system into the pig manure biogas slurry treatment process. This process can purify wastewater and recover resources, making it a carbon-friendly process.

[0019] Fifthly, the present invention provides a wastewater treatment method, which uses the above-mentioned Myco-Algae or the above-mentioned wastewater treatment product to treat wastewater; preferably, the wastewater is biogas slurry from the anaerobic fermentation of pig manure.

[0020] The beneficial effects of this invention are as follows: This invention utilizes low-temperature screening to identify *McClella* species that can grow effectively in low-temperature environments. *McClella* has the CGMCC No. 40696 preservation number. The screened *McClella* can be used in pig manure wastewater with NH4... + It can be grown in environments with -N concentrations higher than 400 mg / L and has good ammonia nitrogen and phosphorus removal effects.

[0021] The *Mexicosporum* species provided by this invention has low-temperature resistance characteristics. As a cold-resistant and stress-resistant algae species that can treat high ammonia nitrogen and high phosphorus pig manure biogas slurry in cold regions, it can reduce the pressure on livestock and poultry wastewater treatment and resource utilization in high-altitude and cold regions. Attached Figure Description

[0022] 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.

[0023] Figure 1 This is an electron microscope image of the culture and electron micrograph of the Mycorrhizal W1 strain of the present invention.

[0024] Figure 2 This is the growth and development tree of the Mycorrhizal Fiber W1 of this invention.

[0025] Figure 3 This is a temperature-comparison growth curve of the *Mycorrhiza* (CGMCC No. 40696) of this invention.

[0026] Figure 4 This is the growth curve of the *Mycorrhiza gracilis* (CGMCC No. 40696) of this invention cultured in biogas slurry at 15℃.

[0027] Figure 5 The results show the ammonia nitrogen removal rate of the biogas slurry culture comparative test of the *Mycorrhiza gracilis* (CGMCC No. 40696) of this invention at 15℃.

[0028] Figure 6 The results show the total phosphorus removal rate of the biogas slurry culture comparative test of the present invention, Mycorrhizal spp. (CGMCC No. 40696), at 15℃. Detailed Implementation

[0029] 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.

[0030] Example 1: Algal strain isolation, purification and culture The algal strain in this embodiment was isolated from soil samples taken from near a biogas digester in a village in Harbin, Heilongjiang Province, which had been contaminated by anaerobic fermentation of pig manure. The soil samples were collected in late April (ambient temperature: 0-20℃). The moist, active soil samples were placed in clean plastic bottles, sealed, and transported to our laboratory via low-temperature circulating express delivery, where they were stored at 4℃ for later use.

[0031] Approximately 0.5 g 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 observed on the plate. A sterile single algal colony was then picked and inoculated into liquid BG11 medium and cultured in a light incubator.

[0032] Table 1 BG11 Culture Medium Formulation

[0033] 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.

[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 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.

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

[0037] Example 2: Algal species identification and preservation During the process of screening algae in cold soils, 16 algae were initially screened from different soil samples. After enrichment, separation and purification, one surviving Mycorrhizal spp. was obtained and named W1.

[0038] When identifying algal strains, preliminary morphological observation is performed first, followed by molecular biological identification.

[0039] 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. 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] (1) Extraction of genomic DNA and PCR amplification 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.

[0042] 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 high-efficiency, high-fidelity enzymes to ensure amplification efficiency and accuracy.

[0043] a) Mixing and purification of PCR products 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.

[0044] b) Library construction and sequencing 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.

[0045] 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 and genus were determined. The growth and development tree is shown below. Figure 2 As shown.

[0046] (2) Preservation of algal strains The *McClella* strain W1, obtained and identified through isolation and purification, was deposited on May 31, 2023, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 40696, and the classification name is *McClella*. Mychonastes sp.

[0047] To verify that the isolated, purified, and preserved algal strain CGMCC No.40696 has cold resistance and stress tolerance 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.

[0048] Example 3: Verification of the cold resistance and stress tolerance of isolated and purified algal strains The culture medium used in the experiment was standard BG11 medium, the formula of which is shown in Table 1. The algal strain used in the experiment was the algal strain purified in the previous isolation and purification experiment (preservation number CGMCC No. 40696). In addition, two control algal strains were introduced, one of which was C-1, *Spirometra spp.* Stichococcus antarticus FACHB-2327, purchased from the Freshwater Algae Culture Bank of the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China), is a low-temperature tolerant algae strain selected from the Fildes Peninsula in Antarctica. Its original designation was UA4, and C-2 represents Chlamydomonas from the laboratory. Chlamydomonas (sp.), with accession number: CGMCC No.15497, is an algal species that grows well at room temperature and has been disclosed in Chinese patent CN109251866B.

[0049] (1) Growth status of algae at different temperatures 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 250 mL Erlenmeyer flasks with a working volume of 150 mL. 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.

[0050] (2) Separation and purification of algal strains to purify pig manure biogas slurry The anaerobic fermentation slurry from pig manure was taken from a biogas digester in a village 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 min. 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℃ 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.

[0051] The algal strain used in the experiment was the one purified in Example 1. It was cultured in BG11 medium until 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; its properties are shown in Table 2.

[0052] Table 2. Water quality characteristics of biogas slurry after 10-fold dilution Water quality indicators Mean ± Standard Deviation COD (mg / L) 1918.72±66.50 <![CDATA[NH4 + -N(mg / L)]]> 406.95±2.95 TP (mg / L) 24.37±0.03 Chromaticity (PCU) 1118.87±30.64 pH 8.20±0.05 The reactor used in the experiment was a 250 mL Erlenmeyer flask with a working volume of 150 mL. 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 200 μmol / m². 2 The photoperiod (L:D) was 12:12, the culture time was 12 days, and the bottles were manually shaken three times a day.

[0053] The test results are as follows: 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, the Microalgae strain CGMCC No.40696 showed good growth under both normal temperature (25℃) and low temperature (15℃) conditions.

[0054] On day 12 under low temperature (15℃) conditions, the biomass of *McClella* strain CGMCC No.40696 reached 257.66 mg / L, the biomass of *Spirometra* strain FACHB-2327 was 253.22 mg / L, and the growth of *Chlamydomonas* strain CGMCC No.15497 was the worst, with a biomass of 227.63 mg / L.

[0055] 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 Mycophylloides 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.

[0056] Comparing the three microalgae, their growth in pig manure biogas slurry at 15℃ is as follows: Figure 4 As shown in the figure, during the 12-day culture period, all three microalgae were able to grow to varying degrees in pig manure biogas slurry. Among them, *Mycorrhiza* strain CGMCC No. 40696 showed the best growth in pig manure biogas slurry, with a biomass of 204.67 mg / L on day 12. *Spirometra hygroscopica* strain FACHB-2327 had a biomass of 191.12 mg / L on day 12 in pig manure biogas slurry. *Chlamydomonas* strain CGMCC No. 15497 showed the worst growth in pig manure biogas slurry, with a biomass of 143.63 mg / L on day 12.

[0057] At a low temperature of 15℃, the doubling time of the *Mexicoplaninus* strain CGMCC No. 40696 in biogas slurry was 2.13 days.

[0058] 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 Mycophylloides strain isolated and purified by this invention has the best tolerance in pig manure biogas slurry and the strongest ability to accumulate biomass.

[0059] 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, *Mycorrhiza* strain CGMCC No. 40696 showed the strongest removal ability of ammonia nitrogen from pig manure biogas slurry, with a removal rate of 44.61% after 12 days; *Spirometra hygroscopica* strain FACHB-2327 showed the second strongest removal ability, with a removal rate of 43.67% after 12 days; and *Chlamydomonas* strain CGMCC No. 15497 showed the weakest removal ability of ammonia nitrogen, with a removal rate of only 42.94% after 12 days.

[0060] 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 6As 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.

[0061] During the 12-day cultivation period, the removal rate of total phosphorus in pig manure biogas slurry was 8.30% for the *McClella* strain CGMCC No. 40696, 5.03% for the *Fragaria hygroscopica* strain FACHB-2327, and the worst removal rate of total phosphorus in pig manure biogas slurry was only 3.15% for the *Chlamydomonas* strain CGMCC No. 15497.

[0062] In summary, the *McClella* strain CGMCC No. 40696 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 *Schizophyllum commune* strains randomly selected from commercial algal banks and *Chlamydomonas* strains previously preserved by our research group, this wild-type algae 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.

[0063] 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. A strain of Mycorrhiza ( Mychonastes sp W1, characterized in that, The preservation number of the *Mycorrhiza* is CGMCC No. 40696.

2. A wastewater treatment product, characterized in that, Contains *McClella* strain or *McClella* liquid; the preservation number of *McClella* is CGMCC No. 40696; The wastewater is biogas slurry produced from the anaerobic fermentation of pig manure wastewater, and the biogas slurry contains NH4. + -N concentration is 400-600 mg / L.

3. The application of the Mycoalac 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 ambient temperature for this application is 15°C.

5. A wastewater treatment method, characterized in that, Wastewater is treated using the Mycoal as described in claim 1 or the wastewater treatment product as described in claim 2. The wastewater is biogas slurry produced from the anaerobic fermentation of pig manure wastewater, and the biogas slurry contains NH4. + -N concentration is 400-600 mg / L.

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