Chlorella for efficiently removing eutrophic elements in sewage and application of chlorella

Abstract

The invention discloses chlorella for efficiently removing eutrophic elements in sewage and application of the chlorella, and relates to the technical field of eutrophic sewage treatment. According to the invention, the chlorella sp. 111-4 is obtained through screening, and can efficiently remove eutrophic elements such as nitrogen, phosphorus and TOC in sewage; the chlorella sp. 111-4 is preserved in the China Center for Type Culture Collection, and the preservation number of the chlorella sp. 111-4 is CCTCC NO: M 2025475. The chlorella sp. 111-4 is inoculated into the sewage to be treated, the sewage treatment effect is more remarkable than that of other reported chlorella, and the degradation rate of nitrogen and phosphorus in the wastewater within 7 days reaches up to 97% or above.

Description

Technical Field

[0001] The present invention relates to the technical field of eutrophic sewage treatment, and particularly relates to a chlorella for efficiently removing eutrophic elements from sewage and its application. Background Art

[0002] Most of the wastewater generated during production, life and other processes contains various harmful substances. If it is directly discharged into the environment without treatment, it will cause water eutrophication and an increase in the content of toxic substances such as heavy metals, which will pose a serious threat to the environment and human health. Among them, water eutrophication refers to the phenomenon of water pollution caused by excessive content of nutrients such as nitrogen and phosphorus in wastewater. This phenomenon has become increasingly serious under the influence of human activities and has become one of the current global environmental problems.

[0003] The sewage treatment processes for eutrophic wastewater include various methods such as physical, chemical and biological methods. Conventional treatment methods such as dredging of water bottom mud, artificial aeration on the water surface, adding chemical agents, diverting water to dilute and reduce the nutrient salt level, etc. Although they can achieve the restoration of eutrophic water bodies in a short time, they may also damage the original ecological environment, and have a large project volume, high construction cost, and are prone to secondary pollution. Generally, they are only used as emergency measures. The biological water quality restoration technology uses the biological absorption, biological transformation and biological accumulation effects of specific organisms on eutrophic elements in water during the metabolic process to reduce the content of eutrophic elements in water, improve water quality, and restore the ecological function of water bodies. The biological water quality restoration technology has attracted much attention due to its low investment, high benefit, easy application and great development potential.

[0004] Water eutrophication is often accompanied by a large growth of algae, resulting in algal blooms, which have a negative impact on the ecological environment. It is worth noting that the growth of algae itself is a natural form of fixing and removing eutrophic elements in water. If this characteristic of algae can be applied and a process technology is established to safely transfer and fix the nitrogen and phosphorus elements in algal biomass, an effective algal-based bioremediation technology can be formed. In fact, using algae to achieve biological treatment of eutrophic wastewater has become a new trend in sewage treatment technology. Algal cells can use inorganic and organic nitrogen compounds as nitrogen sources, such as nitrates, nitrites and ammonium salts, etc. These nitrogen sources can be used by algae to synthesize amino acids and proteins. At the same time, after phosphate is absorbed by algal cells, it is converted into organic substances such as ATP and phospholipids through various phosphorylation pathways, thereby achieving the removal of phosphorus. The technology of using algae to remove nitrogen and phosphorus from wastewater has been widely applied in various wastewater treatment scenarios such as aquaculture wastewater and urban domestic sewage.

[0005] Although the technology of using algae to remove nitrogen and phosphorus from wastewater has broad application prospects, it still faces many challenges. Especially at the technical level, the selection and breeding of functional algal species with high-efficiency nitrogen and phosphorus removal are not perfect. The algae obtained by purification and separation in nature often have low removal efficiency and are difficult to adapt to high-concentration sewage environments. There are various reasons for this problem, for example:

[0006] ① Limited germplasm resources: Although there are a large number of different algae in nature, not all algae have the ability to efficiently remove nitrogen and phosphorus. At the same time, the current exploration of algal species resources with high-efficiency nitrogen and phosphorus removal ability is insufficient, resulting in a limited range of choices in the selection and breeding process.

[0007] ② High screening difficulty: Screening out algal species with high-efficiency nitrogen and phosphorus removal ability from a large number of algae requires a lot of time and resources. At the same time, a scientific evaluation system needs to be established during the screening process to accurately evaluate the removal efficiency and adaptability of different algal species.

[0008] ③ Poor genetic stability: Some algal species may undergo genetic variation during long-term domestication and cultivation, resulting in a decrease in removal efficiency. Poor genetic stability may also cause the algal species to be difficult to maintain a stable removal effect in practical applications.

[0009] ④ Challenges of adaptability: The types and concentrations of pollutants in wastewater are complex and variable, and the water quality differences between different wastewaters are significant. Algal species need to have good adaptability and be able to maintain high-efficiency removal efficiency under different water quality conditions.

[0010] ⑤ Challenges in other aspects: The growth and removal efficiency of algal species are affected by various environmental factors such as light, temperature, pH value, and nutrient concentration. Fluctuations in environmental factors may lead to limited growth of algal species or a decrease in removal efficiency. In a wastewater treatment system, there may be other microorganisms or algae competing with the selected algal species for nutrients and living space, and this competition relationship may inhibit the growth of the selected algal species, thus affecting its removal efficiency.

[0011] All in all, algae are naturally distributed in various eutrophic water bodies. Using algae to remove nitrogen and phosphorus elements from eutrophic water bodies has great application prospects. However, there is currently a lack of algal species resources with strong water quality adaptability, short growth cycles, fast metabolic rates, low breeding costs, and high nitrogen and phosphorus removal efficiency. Summary of the Invention

[0012] The present invention provides a Chlorella sp. and its application for efficiently removing eutrophic elements from sewage. Through a complete set of methods including field sampling, algal species isolation and identification, algal species cultivation, verification of nitrogen and phosphorus element removal, and sewage treatment test methods, a Chlorella sp. capable of removing eutrophic elements such as nitrogen (mainly ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, etc.), phosphorus (mainly orthophosphate), and organic matter from sewage has been screened out. This Chlorella sp. has the advantages of strong adaptability to sewage quality, fast amplification rate, high nitrogen and phosphorus removal efficiency, etc. It is specifically realized through the following technologies.

[0013] The present invention provides an application of Chlorella sp. 111-4 in efficiently removing eutrophic elements from sewage.

[0014] Furthermore, the Chlorella sp. is inoculated into the sewage to be treated for the degradation of nitrogen, phosphorus, and TOC.

[0015] The present invention also provides a method for efficiently removing eutrophic elements from sewage, wherein Chlorella sp. 111-4 is inoculated into the sewage to be treated for the degradation of nitrogen, phosphorus, and TOC; the Chlorella sp. 111-4 was deposited at the China Center for Type Culture Collection on March 14, 2025, with the deposit number CCTCC NO: M2025475 and the deposit address being Wuhan University, Wuhan, China.

[0016] Furthermore, in the sewage to be treated, the original contents of total nitrogen, total phosphorus, and TOC do not exceed 120.885 mg / L, 14.39 mg / L, and 69.545 mg / L respectively.

[0017] Furthermore, based on the volume of the sewage to be treated, the inoculation amount of Chlorella sp. 111-4 is 0.17 mg / L.

[0018] Furthermore, the conditions for the degradation are: a temperature of 22 °C, a light intensity of 30 μmol / (m 2 ∙s) with continuous illumination, and air containing 4% carbon dioxide by volume filtered through a 0.22 μm membrane is introduced.

[0019] Furthermore, the eutrophic elements are nitrogen, phosphorus, and TOC.

[0020] Furthermore, before inoculating the Chlorella sp. 111-4, the sewage to be treated is pretreated to remove particulate impurities and / or adjust the pH value.

[0021] Further, after removing the eutrophic elements in the wastewater to be treated, Chlorella sp. 111-4 in the water body is separated and recovered, dried, and the biological oil and / or protein in Chlorella sp. 111-4 is extracted, or it is used as an organic fertilizer.

[0022] Generally, Chlorella sp. 111-4 can be separated from wastewater by appropriate harvesting methods (such as flocculation, sedimentation, filtration, centrifugation, etc.) for subsequent treatment and disposal; it can avoid secondary pollution and improve the biological recycling of Chlorella sp. 111-4. For example, useful components such as biological oil and protein in Chlorella sp. 111-4 can be extracted, and Chlorella sp. 111-4 can also be used as a biological organic fertilizer or animal feed.

[0023] The present invention also provides a Chlorella sp. 111-4, which is characterized in that the Chlorella sp. 111-4 was deposited with the China Center for Type Culture Collection on March 14, 2025, with the deposit number CCTCC NO: M 2025475 and the deposit address being Wuhan University, Wuhan, China; the Chlorella sp. 111-4 is used for efficiently removing eutrophic elements in wastewater.

[0024] The Chlorella sp. 111-4 provided by the present invention is an in-situ cultured alga separated, screened and cultured in treatment units such as the aeration tank, sedimentation tank or biological reaction tank of a wastewater treatment plant, and has the ability to efficiently degrade nitrogen, phosphorus and TOC.

[0025] Generally speaking, factors such as algal species selection, light conditions, temperature and pH, nutrients and hydraulic retention time are the keys affecting the efficiency of algae in removing eutrophic elements in water bodies. By optimizing the growth conditions of the Chlorella sp. 111-4 of the present invention, for example, adjusting parameters such as light, temperature, pH value, concentration of nutrients (such as nitrogen, phosphorus, carbon source, etc.) in the wastewater and the aeration volume, the growth environment of the Chlorella sp. 111-4 is optimized, and the absorption efficiency of nitrogen and phosphorus can be significantly improved.

[0026] Among them, light is an important influencing factor for algal photosynthesis. In the present invention, under appropriate light time and light intensity (such as a light-dark contrast of 12h:12h and a light intensity of 4000 lux), the removal rate of ammonia nitrogen in wastewater by Chlorella sp. 111-4 can reach a relatively high level.

[0027] Chlorella sp. 111-4 also has a certain adaptability to pH, but it usually grows better under neutral conditions; too high or too low pH may have an adverse effect on the growth of algae.

[0028] Temperature affects the growth and development of algae by influencing the activity of enzymes and the absorption and utilization of nutrients. Chlorella sp. 111-4 provided by the present invention has the best growth and nitrogen and phosphorus removal effects within a suitable temperature range (such as 24-28 °C).

[0029] The concentration and composition of nutrients (TOC) play an important role in the growth and development of algae. There are differences in the ammonia nitrogen removal rates of Chlorella sp. 111-4 under autotrophic and heterotrophic modes.

[0030] The nitrogen-phosphorus ratio also affects the phosphorus absorption rate.

[0031] The sludge retention time has a great influence on the water treatment efficiency of Chlorella sp. 111-4. An appropriate sludge retention time helps to maintain the stable growth of algae in the system and a high nitrogen and phosphorus removal rate. By utilizing the characteristics of algae naturally absorbing nitrogen (including ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, etc.) and phosphorus (mainly orthophosphate) in sewage during the growth process, the nitrogen and phosphorus removal rate can be further improved by extending the contact time between sewage and algae and increasing the algal biomass.

[0032] (5) Water quality monitoring and process adjustment: Monitor the treated water quality parameters every day, including but not limited to nitrogen and phosphorus concentrations, chemical oxygen demand (COD), and the growth status of algae, and adjust the treatment process parameters in a timely manner according to the monitoring results to ensure the stability and optimization of the nitrogen and phosphorus removal efficiency.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1. The Chlorella provided by the present invention has a more significant sewage treatment effect than other reported Chlorella in terms of degrading total nitrogen, total phosphorus, and TOC. The nitrogen and phosphorus degradation rate in wastewater is as high as over 97% within 7 days, and it is an in-situ alga with high water purification efficiency.

[0035] 2. The Chlorella provided by the present invention can well adapt to the in-situ water quality of sewage treatment plants and has an extremely fast growth rate. Description of the Drawings

[0036] Figure 1 It is a diagram of the algal species collection process.

[0037] Figure 2It is a flow chart for the in-situ algae separation, purification and screening.

[0038] Figure 3 It is a graph showing the growth status of in-situ algae in BG11 medium.

[0039] Figure 4 It is a graph showing the operation of the microalgae sewage treatment system device.

[0040] Figure 5 It is a graph showing the growth status of in-situ algae in wastewater.

[0041] Figure 6 It is a graph showing the removal effect of in-situ algae on TN (total nitrogen) in wastewater.

[0042] Figure 7 It is a graph showing the removal effect of in-situ algae on TP (total phosphorus) in wastewater.

[0043] Figure 8 It is a graph showing the removal effect of in-situ algae on TOC (total organic carbon) in wastewater. Specific implementation mode

[0044] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0045] Example 1: Collection, separation and purification of algae for efficient removal of eutrophic elements in sewage

[0046] The specific method is as follows:

[0047] 1. Collection, separation and purification of in-situ algae in sewage treatment plants

[0048] (1) As Figure 1 shown, use a scraper to collect algae and microbial symbiotic moss on the water side of the sewage treatment plant, drag the surface layer to a depth of 0.5 m with a No. 25 plankton net (mesh size 64 μm), and place the enriched fresh algal liquid in a 10 mL centrifuge tube, and store it at low temperature and bring it back to the laboratory for selection and separation.

[0049] (2) After the number of cells increases, separate and gradually purify the microalgae from the brought-back wastewater and its sludge through the dilution pour plate and plate separation techniques, and use the capillary separation method to separate the experimental algal species.

[0050] The specific separation method is:

[0051] ① Heat one end of the capillary with an alcohol lamp, and then when it melts, quickly stretch the heated end 6 - 10 cm with forceps to reduce the diameter.

[0052] ②Use forceps to clamp and break at the narrowed part, and pick up the algal species with the narrowed end of the capillary tube.

[0053] ③Drop a drop of water sample in the middle of a glass slide and observe it under an inverted microscope. While blocking one end of the capillary tube with the right index finger, slowly insert the narrowed end into the sample. Align the tip of the capillary tube with the alga to be separated, release the right index finger, and suck the alga into the capillary tube using the siphon method.

[0054] ④Transfer the alga in the capillary tube to a drop of sterilized BG11 medium for washing, and separate it again with the capillary tube. After washing four to five times, transfer it to a 96-well plate filled with BG11 medium and culture it in an artificial incubator. After 10 - 15 days, conduct microscopic examination. If the alga in the culture plate is not pure, it needs to be separated again until a single target algal species appears.

[0055] The BG-11 medium used in the above experiment has the formula shown in Tables 1 and 2 below.

[0056] Table 1 BG11 Medium Formula

[0057]

[0058] Table 2 Formula of Reagent A5 in BG11 Medium Formula

[0059]

[0060] Solid medium: Add 1.5% agar to the above BG11 liquid medium. After sterilization, in a laminar flow hood, add about 15 - 20 mL of the medium to each petri dish. After it solidifies, seal it with a film for standby.

[0061] 2. Screening and Amplification Culture of Functional Algal Species

[0062] (1) When the alga in the cell culture plate grows to a certain concentration, transfer it to the BG11 solid medium and continue to culture for 5 - 8 days.

[0063] (2) Pick the well-growing and uncontaminated algal colonies from the petri dish and inoculate them into a small amount of BG11 liquid medium for continuous culture.

[0064] (3) When it grows to a certain concentration, inoculate it into a 250 mL conical flask for large-scale culture. The culture conditions are: 25 °C, light intensity 30 μmol / (m 2 ·s), under a 12 h / 12 h light-dark cycle, and culture it in an incubator.

[0065] It should be noted that to make the algal species grow evenly, it is necessary to shake regularly during the culture process.

[0066] (4)Liquid preservation: The algal strains were preserved by the method of low temperature and weak light. Specifically, 120 mL of sterilized BG11 medium (formula shown in Table 1) was added to a sterilized 250 mL conical flask, 10 mL of algal liquid in the stable growth phase was inoculated, and the name of the algal strain and the date were marked. Then it was placed in an illumination incubator for cultivation.

[0067] The cultivation conditions were: light intensity 30 μmol / (m 2 ·s), temperature 15 °C, and light-dark ratio 12:12. After three weeks of cultivation, transfer was carried out in the same way, and microscopic examination was often carried out to prevent contamination.

[0068] (5)The in-situ algal cells after expansion culture were collected and resuspended in the collected raw wastewater, and then cultivated using an aerated photobioreactor on a light shelf.

[0069] The cultivation conditions were: 22 °C, light intensity 30 μmol / (m 2 ∙s), continuous illumination, and air containing 4% (v / v) carbon dioxide filtered through a 0.22 μm filter membrane was introduced to make it grow.

[0070] (6)Samples were taken once a day for 7 days. The accumulation of algal dry weight biomass and the consumption of nitrogen and phosphorus in the water body were measured respectively. The basic data were sorted out, and the growth curves, nitrogen and phosphorus removal curves, and TOC removal curves of each in-situ alga were drawn.

[0071] (7)Based on the growth curves, nitrogen and phosphorus removal curves, and TOC removal curves of each in-situ alga above, a functional algal strain with a fast growth rate and high nitrogen and phosphorus removal efficiency was screened out.

[0072] (8)Morphological analysis was carried out on the screened functional algal strain, the genome of the functional algal strain was extracted, PCR amplification was carried out and gene sequence sequencing was performed, and the biological classification of the functional algal strain was identified by molecular biology. Finally, it was found that the functional algal strain belongs to Chlorella sp., and it was named Chlorella sp. 111-4.

[0073] The screened functional algal strain "Chlorella sp. 111-4" was deposited at the China Center for Type Culture Collection on March 14, 2025, with the deposit number CCTCC NO: M 2025475, and the deposit address is Wuhan University, Wuhan, China.

[0074] Example 2: Test on the degradation and removal effect of nitrogen and phosphorus elements in the water body of a sewage treatment plant by a functional algal strain

[0075] The operation diagram of the microalgae sewage treatment system device is as Figure 5 shown, and the specific test method is as follows:

[0076] (1) Add 120 mL of sterilized BG11 medium (formula shown in Table 1) into a sterilized 250 mL conical flask, inoculate 10 mL of Chlorella vulgaris algal solution in the stable growth phase, label the algal species name and date, and place it in an illumination incubator for cultivation. After two weeks of cultivation, transfer it according to the same method and frequently check under the microscope to prevent contamination.

[0077] The cultivation conditions are: light intensity 30 μmol / (m 2 ·s), temperature 22 °C, light intensity 30 μmol / (m 2 ∙s).

[0078] (2) Take 100 mL of the enriched algal solution, centrifuge it at 3000 rpm for 7 min, discard the supernatant, and collect the algal bodies. Suspend the algal bodies with 10 mL of wastewater and inoculate them into a 2000 mL Erlenmeyer flask containing 1500 mL of wastewater. Calculated based on the volume of the wastewater to be treated and the dry weight of Chlorella vulgaris, the inoculation amount is approximately 0.17 mg / L. Cultivate it in an aerated photobioreactor on an illumination rack, set the cultivation conditions as 22 °C, light intensity 30 μmol / (m 2 ∙s), continuously irradiate with light and introduce air containing 4% (v / v) carbon dioxide filtered through a 0.22 μm filter membrane to make it grow.

[0079] (3) Take samples daily for a total of seven days. Measure its OD 680nm , and use the potassium persulfate oxidation-ultraviolet spectrophotometry and potassium persulfate oxidation-ultraviolet spectrophotometry to measure the accumulation of algal dry weight biomass and the consumption of nitrogen and phosphorus in the water body respectively. Plot the algal growth curve and nitrogen and phosphorus removal curves with the measured data, as shown in Figures 5 - 8 respectively.

[0080] It can be seen that during the cultivation period, Chlorella vulgaris continuously absorbs and utilizes the nitrogen and phosphorus nutrients in the wastewater, the concentrations of nitrogen and phosphorus substances in the wastewater decrease, and the removal rate gradually increases. Calculated based on the total nitrogen (TN), total phosphorus (TP), and total organic carbon (TOC) in the water body of the sewage treatment plant, the content of total nitrogen decreased from 120.89 mg / L to 1.22 mg / L within 7 days, and the degradation rate was as high as over 97%. The content of total phosphorus decreased from 14.39 mg / L to 0.26 mg / L, and the degradation rate was as high as 98%. The content of total organic carbon decreased from 69.6 mg / L to 17.8 mg / L, and the degradation rate reached 74%.

[0081] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple variations all fall within the protection scope of the present invention.

Claims

1. An application of Chlorella sp. 111-4 in the efficient removal of eutrophic elements in sewage.

2. The use of Chlorella sp. 111-4 in efficiently removing eutrophic elements from sewage according to claim 1, characterized in that: Chlorella is inoculated into the wastewater to be treated to degrade nitrogen, phosphorus and TOC.

3. A method for efficiently removing eutrophic elements from sewage, characterized in that: Chlorella sp. 111-4 is inoculated into the sewage to be treated to degrade nitrogen, phosphorus and TOC; the Chlorella sp. 111-4 has been deposited in the China Center for Type Culture Collection on March 14, 2025, with a deposit number of CCTCC NO: M 2025475, and the deposit address is Wuhan University, Wuhan, China.

4. The method for efficiently removing eutrophic elements from sewage according to claim 3, characterized in that: In the sewage to be treated, the original contents of total nitrogen, total phosphorus and TOC do not exceed 120.885 mg / L, 14.39 mg / L and 69.545 mg / L, respectively.

5. The method for efficiently removing eutrophic elements from sewage according to claim 3, characterized in that: Based on the volume of the sewage to be treated, the inoculation amount of the Chlorella sp. 111-4 is 0.17 mg / L.

6. The method for efficiently removing eutrophic elements from sewage according to claim 3, characterized in that: The degradation conditions are as follows: temperature is 22°C, light intensity is 30 μmol / (m 2 ∙s) were continuously illuminated and air containing 4% carbon dioxide by volume filtered through a 0.22 μm filter membrane was introduced.

7. The method for efficiently removing eutrophic elements from sewage according to claim 3, characterized in that: The nutrient-rich elements are nitrogen, phosphorus and TOC.

8. The method for efficiently removing eutrophic elements from sewage according to claim 3, characterized in that: Before inoculating the Chlorella sp. 111-4, the wastewater to be treated is pretreated to remove particulate impurities and / or adjust the pH value.

9. The method for efficiently removing eutrophic elements from sewage according to claim 3, characterized in that: After the eutrophic elements in the wastewater to be treated are removed, the Chlorella sp. 111-4 in the water is separated and recovered, dried, and the bio-oil and / or protein in the Chlorella sp. 111-4 is extracted or used as an organic fertilizer or animal feed.

10. A Chlorella sp. 111-4, characterized in that: The Chlorella sp. 111-4 was deposited in the China Center for Type Culture Collection on March 14, 2025, with a deposit number of CCTCC NO: M2025475, and a deposit address of Wuhan University, Wuhan, China; the Chlorella sp. 111-4 is used to efficiently remove eutrophic elements in sewage.

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