A method for immobilizing Chlorella and its application in removing nitrogen and phosphorus from aquaculture wastewater

Abstract

This invention relates to a method for immobilizing Chlorella, in order to... Chlorella Using *Chlorella sp. FACHB-9* as the culture medium, *Chlorella* solution was inoculated into the culture medium and cultured under conditions where light intensity varied with concentration to obtain a high-concentration *Chlorella* solution. Chitosan-modified rope-shaped artificial aquatic plants, used as an immobilization carrier, were added to the high-concentration *Chlorella* solution and fixed in a constant-temperature shaker at 25–35℃ and 120–180 rpm for 20–30 hours. After removal and drainage, immobilized *Chlorella* was obtained. An immobilized *Chlorella* was prepared by the above method. An application of the immobilized *Chlorella* was also described: the immobilized *Chlorella* was used to remove nitrogen and phosphorus from aquaculture wastewater. The beneficial effects included: effective removal of nitrogen and phosphorus pollution from aquaculture wastewater, prevention of eutrophication, reduction of wastewater treatment costs, inhibition of *Microcystis aeruginosa* growth, and creation of additional economic value through the resource utilization of *Chlorella*.

Description

Technical Field

[0001] This invention relates to the field of water environment pollution control technology, specifically to a method for immobilizing Chlorella and its application in removing nitrogen and phosphorus from aquaculture wastewater. Background Technology

[0002] The rapid development of aquaculture has led to the discharge of large amounts of nitrogen- and phosphorus-rich wastewater, which can easily cause eutrophication, algal blooms, and ecological imbalance. Currently common methods for treating aquaculture wastewater include physical sedimentation, chemical sedimentation, and biological nitrogen and phosphorus removal. However, these methods often suffer from high costs, inconsistent treatment effectiveness, or the potential for secondary pollution. Therefore, developing an efficient, low-cost, and environmentally friendly aquaculture wastewater treatment technology is of great significance for water environment protection.

[0003] Chlorella ( Chlorella Chlorella sp. is a photosynthetic autotrophic microalga that can efficiently absorb nitrogen and phosphorus from water and convert them into its own biomass through biosynthesis, thus achieving resource utilization. Therefore, using Chlorella sp. to treat aquaculture wastewater can not only effectively remove pollutants but also generate biomass resources with high economic value, showing broad application prospects. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for immobilizing Chlorella and its application in removing nitrogen and phosphorus from aquaculture wastewater, so as to overcome the shortcomings of the prior art.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A method for immobilizing Chlorella vulgaris includes the following steps: S10, with Chlorella Using sp. FACHB-9 Chlorella as the culture object, Chlorella in the logarithmic growth phase was inoculated into the culture medium under aseptic conditions and cultured for 4 to 7 days at a temperature of 22℃ to 32℃ with light intensity varying with concentration to obtain high-concentration Chlorella solution. S20. Add the chitosan-modified rope-shaped artificial aquatic plants, which serve as the immobilization carrier, to a high-concentration Chlorella solution and fix it in a constant-temperature shaker at 25℃~35℃ and 120rpm~180rpm for 20h~30h. Then remove it and drain the water to obtain immobilized Chlorella.

[0006] The beneficial effects of this invention are: by optimizing the culture conditions of Chlorella sp. FACHB-9, it ensures... ChlorellaSp. FACHB-9 Chlorella can grow efficiently in aquaculture wastewater environments, improving the absorption efficiency of nitrogen and phosphorus in the water, thus effectively removing nitrogen and phosphorus pollution from aquaculture wastewater, preventing eutrophication, and reducing wastewater treatment costs. This achieves ecological treatment of aquaculture wastewater through optimization. Chlorella High-concentration Chlorella solution was obtained by cultivating sp. FACHB-9 Chlorella. When this solution was combined with chitosan-modified rope-like artificial aquatic plants... Chlorella The biomass attachment rate (based on the dry weight of algal cells attached per unit length of carrier) of sp. FACHB-9 Chlorella cells on the surface of chitosan-modified rope-shaped artificial aquatic plants is 35% to 50% higher than that of ordinary Chlorella, thus enhancing the purification effect. Immobilized Chlorella can inhibit the growth of Microcystis thunbergii.

[0007] Based on the above technical solution, the present invention can be further improved as follows.

[0008] Furthermore, by culturing at 25°C for 5 days under conditions where light intensity varied with concentration, a high-concentration Chlorella solution was obtained.

[0009] Furthermore, the culture medium was BG11 medium at a concentration of 2.5 to 3.5 times.

[0010] Furthermore, the culture medium was 3 times the concentration of BG11 medium.

[0011] Furthermore, the BG11 medium comprises: 1.50 g sodium nitrate, 0.04 g dipotassium hydrogen phosphate, 0.075 g magnesium sulfate heptahydrate, 0.036 g calcium chloride, 0.020 g sodium carbonate, 0.006 g citric acid, 0.006 g ferric ammonium citrate, 0.001 g disodium EDTA, 2.86 mg boric acid, 1.86 mg manganese chloride tetrahydrate, 0.222 mg zinc sulfate heptahydrate, 0.39 mg sodium molybdate dihydrate, 0.079 mg copper sulfate pentahydrate, 0.049 mg cobalt nitrate hexahydrate, and 1000 mL distilled water.

[0012] Furthermore, the preparation method of BG11 medium is as follows: mix all components and adjust the pH to 7.1-7.5, then sterilize at 121°C for 20 minutes in an autoclave.

[0013] Furthermore, under aseptic conditions, Chlorella syriacus solution in the logarithmic growth phase was inoculated into the culture medium at an inoculation rate of 1% to 10%.

[0014] Furthermore, under aseptic conditions, 1% of Chlorella vulgaris solution in the logarithmic growth phase was inoculated into the culture medium.

[0015] Furthermore, the light intensity is: 80 μmol / m 2 / s~150μmol / m 2 / s.

[0016] Further, after fixing in a constant temperature shaker at 30℃ and 150rpm for 24 hours, remove and drain the water.

[0017] Based on the above technical solution, the present invention also provides an immobilized Chlorella prepared by the above method.

[0018] Based on the above technical solution, the present invention also provides an application of immobilized Chlorella, which is used to remove nitrogen and phosphorus from aquaculture wastewater.

[0019] The further beneficial effects of adopting the above methods are: it can effectively remove nitrogen and phosphorus pollution from aquaculture wastewater, prevent eutrophication of water bodies, and reduce wastewater treatment costs, thus achieving ecological treatment of aquaculture wastewater.

[0020] Furthermore, the inoculation concentration of immobilized Chlorella was 1 g / L to 3 g / L.

[0021] Furthermore, the algal sludge obtained after the aquaculture wastewater treatment is completed can be developed into aquatic feed, fertilizer, bioenergy, or health products.

[0022] The further beneficial effects of adopting the above are as follows: After the aquaculture wastewater is treated, Chlorella will form a biomass with high protein, high fat and high pigment content after absorbing nitrogen and phosphorus from the fish farming wastewater. Therefore, after the aquaculture wastewater is treated, the algal sludge can be recovered by flocculation sedimentation, centrifugation or membrane filtration. The collected algal sludge can be processed by pressure filtration or freeze drying to obtain algal powder. The algal powder can be used to develop aquatic feed, fertilizer, bioenergy (such as biodiesel) or health products, forming a sustainable industrial chain that combines wastewater treatment and resource utilization, providing reliable technical support for the sustainable development of the aquaculture industry and increasing economic value. Attached Figure Description

[0023] Figure 1 for Chlorella A bar chart showing the removal rates of NH3-N, TN, and TP by sp. FACHB-9 Chlorella in aquaculture wastewater. Figure 2 This graph shows the change in dry weight of Chlorella cells with culture time in A1–A4 media. Figure 3 A bar chart showing the removal rates of ammonia nitrogen, total nitrogen, and total phosphorus by immobilized Chlorella; Figure 4 This is an application diagram of immobilized Chlorella. Detailed Implementation

[0024] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0025] The following steps were taken to screen for Chlorella species with strong adaptability and high nitrogen and phosphorus removal capacity to ensure efficient growth of Chlorella in aquaculture wastewater environments and improve the stability of its decontamination treatment: Select Chlorella sp. FACHB-9 Chlorella Chlorella sp. FACHB-1227 Chlorella and Chlorella kaempferi ( Parachlorella (kessleri), among which, three species of Chlorella were obtained from the freshwater algae strain bank of the Institute of Hydrobiology, Chinese Academy of Sciences. The three Chlorella species were then inoculated into BG11 medium and cultured to OD. 600 The value was approximately 0.25. The inoculum was then transferred to aquaculture wastewater filtered through a 200-mesh screen and incubated at 25°C for 72 hours, with OD measured every 24 hours. 540 The maximum specific growth rate (μ) and the final concentration at 72 h (expressed as cell turbidity concentration) were measured. Under the same aquaculture wastewater culture conditions, the OD values ​​of the algal strains were continuously measured. 540 The specific growth rate (μ) value was calculated by selecting data from the logarithmic growth phase. The judgment logic was: the larger the specific growth rate (μ) value, the faster the cell division and the stronger the proliferation ability of the algal strain in the wastewater, that is, the stronger the adaptability to the wastewater environment. Thus, the removal effect of Chlorella on soluble ammonia nitrogen, total nitrogen and total phosphorus in aquaculture wastewater was measured, so as to screen Chlorella species with strong adaptability and high nitrogen and phosphorus removal capacity.

[0026] The results showed Chlorella sp. FACHB-9 *Chlorella* can rapidly adapt to and proliferate in aquaculture wastewater, and its specific growth rate (μ) is significantly higher than that of the other two microalgae. By measuring the removal effects of the three microalgae on soluble ammonia nitrogen, total nitrogen, and total phosphorus in aquaculture wastewater, it was found that… Chlorella sp. FACHB-9 Chlorella removed NH3-N, TN, and TP at rates of 74.5%, 78.1%, and 83.8%, respectively. Figure 1 As shown, it is higher than the other two microalgae. Through the above screening process, it is considered that... Chlorella sp. FACHB-9 Chlorella has a strong ability to remove nitrogen and phosphorus, making it suitable for research on nitrogen and phosphorus removal from aquaculture wastewater.

[0027] Growth of three species of Chlorella in aquaculture wastewater The BG11 medium consists of: 1.50 g sodium nitrate, 0.04 g dipotassium hydrogen phosphate, 0.075 g magnesium sulfate heptahydrate, 0.036 g calcium chloride, 0.020 g sodium carbonate, 0.006 g citric acid, 0.006 g ferric ammonium citrate, 0.001 g disodium EDTA, 2.86 mg boric acid, 1.86 mg manganese chloride tetrahydrate, 0.222 mg zinc sulfate heptahydrate, 0.39 mg sodium molybdate dihydrate, 0.079 mg copper sulfate pentahydrate, 0.049 mg cobalt nitrate hexahydrate, and 1000 mL distilled water.

[0028] The preparation method of BG11 medium is as follows: mix all components and adjust the pH to 7.1-7.5, and then sterilize at 121℃ for 20 minutes in an autoclave.

[0029] Prepare four culture media, A1 to A4. Media A1 is BG11 medium; Media A2 is BG11 medium at 2x concentration; Media A3 is BG11 medium at 2.5–3.5x concentration; Media A4 contains nitrogen and phosphorus at 4 times the concentration of BG11 medium, and other elements at 2 times the concentration of BG11 medium; as a preferred option, Media A3 is BG11 medium at 3x concentration. Under aseptic conditions, inoculate each medium at an inoculum of 1%–10% of the culture medium in the logarithmic growth phase. Chlorella sp. FACHB-9 Chlorella solution was added to A1–A4 media and cultured for 4–7 days at temperatures of 22–32°C, with light intensity varying with concentration. The light intensity was 80 μmol / m². 2 / s~150μmol / m 2 / s, respectively, to determine its maximum specific growth rate, algal cell density, and algal cell dry weight, such as Figure 2 As shown; The above experiments show that in A3 culture medium... Chlorella The maximum specific growth rate, algal cell density, and algal cell dry weight of sp. FACHB-9 Chlorella reached their optimal values, at 0.784, 1.4 × 10⁻⁶, and 1.4 × 10⁻⁶, respectively. 11 The concentration of ind / L and the dry weight reached 3.45 g / L.

[0030] A method for immobilizing Chlorella vulgaris includes the following steps: S10, with Chlorella Sp. FACHB-9 Chlorella was used as the culture medium. Under aseptic conditions, 1% of the Chlorella solution in the logarithmic growth phase was inoculated into A3 medium. The medium was cultured for 4–7 days at 22℃–32℃ with varying light intensity (80 μmol / m²). 2 / s~150μmol / m 2 / s, a high concentration of Chlorella solution was obtained with a dry weight of 3.45 g / L; S20. Add the chitosan-modified rope-shaped artificial aquatic plants, which serve as the immobilization carrier, to a high-concentration Chlorella solution and fix it in a constant-temperature shaker at 25℃~35℃ and 120rpm~180rpm for 20h~30h. Then remove it and drain the water to obtain immobilized Chlorella.

[0031] A3 culture medium can be dispensed into 2L Erlenmeyer flasks, 1000mL per flask; the main material of the chitosan-modified rope-shaped artificial aquatic plant is polyester, with polypropylene as the support thread, each thread can be 10cm long, or other sizes, this is just an example.

[0032] An application of immobilized Chlorella, wherein the immobilized Chlorella is used to remove nitrogen and phosphorus from aquaculture wastewater, wherein the inoculum concentration of the immobilized Chlorella is 1 g / L to 3 g / L.

[0033] Application Example 1 like Figure 4 As shown, the effects of immobilized Chlorella and non-immobilized Chlorella (high-concentration Chlorella solution, as the control group) on nitrogen and phosphorus removal in aquaculture wastewater and their inhibitory effect on Microcystis aeruginosa were verified. 1) Wastewater from the indoor laboratory of the teaching and internship base of the College of Fisheries, Huazhong Agricultural University was collected, allowed to stand for 1 hour, and then filtered through a 250-mesh plankton net. The filtered water was used as experimental water, and the total ammonia nitrogen was measured to be 0.5 mg·L⁻¹. -1 ~0.6 mg·L -1 The total nitrogen was 2.1 mg·L. -1 ~2.3 mg·L -1 The total phosphorus content was 0.45 mg·L⁻¹. -1 ~0.6 mg·L -1 ; 2) Nitrogen and phosphorus removal experiment of Chlorella: Filtered fish farming wastewater was used as experimental water. The inoculation concentration gradients (by dry weight) of immobilized and non-immobilized Chlorella were 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, and 3 g / L, respectively. Wastewater without Chlorella was used as a control. After culturing for 5 to 7 days, the growth of chlorophyll a and its removal rate of ammonia nitrogen, total nitrogen, and total phosphorus in the wastewater were detected, and its inhibitory effect on Microcystis aeruginosa was observed.

[0034] The results showed that both immobilized and non-immobilized Chlorella could adapt well to the fish farming wastewater environment and inhibit the growth of Microcystis aeruginosa. Among them, the non-immobilized Chlorella showed the largest increase in chlorophyll a concentration at an inoculum concentration of 2 g / L, reaching 1.3 times the initial inoculum. However, under the same hydraulic retention time, the removal rates of ammonia nitrogen, total nitrogen, and total phosphorus by immobilized Chlorella remained stable within the ranges of 75%–85%, 78%–88%, and 82%–92%, respectively (typical values: 77.5%, 80.1%, and 85.3%). Compared to non-immobilized Chlorella (free Chlorella), immobilized Chlorella not only achieved a breakthrough in absolute removal rates, but also... Figure 3 As shown, it is stronger than non-immobilized Chlorella. More importantly, it exhibits extremely strong resistance to shock loads during the treatment process and is less affected by water quality fluctuations. Compared with non-immobilized Chlorella (control group), which requires a large amount of energy for centrifugation or flocculation recovery after wastewater treatment, this invention achieves in-situ fixation and rapid extraction of Chlorella by modifying rope-like artificial aquatic plants with chitosan. After treatment, only the artificial aquatic plants need to be removed to achieve more than 95% algal biomass recovery, which greatly reduces the overall cost of wastewater treatment.

[0035] After the aquaculture wastewater is treated, Chlorella absorbs nitrogen and phosphorus from the fish farming wastewater and forms a biomass with high protein, high fat and high pigment content. The resulting algal sludge can be developed into aquatic feed, fertilizer, bioenergy (such as biodiesel) or health products, forming a sustainable industrial chain that combines wastewater treatment and resource utilization, thereby increasing economic value.

[0036] For example, since Chlorella absorbs nitrogen and phosphorus from fish farming wastewater and forms biomass with high protein, high fat, and high pigment content, after the aquaculture wastewater treatment is completed, the algal sludge is recovered through flocculation sedimentation, centrifugation, or membrane filtration. The collected algal sludge is then processed by pressure filtration or freeze drying to obtain algal powder, the typical components of which are: protein content of 35%–55%, lipid content of 15%–30%, and chlorophyll and carotenoid content of 0.5%–2.5%. The algal powder is added to the formulated feed at a ratio of 5%–20%, and after mixing, granulation, and drying, an aquatic feed rich in algal protein and natural pigments is obtained. Feeding experiments show that this feed can increase the growth rate of fish by 8%–15% and improve the color and immunity of fish.

[0037] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for immobilization of Chlorella sp. characterized by, Includes the following steps: S10, with Chlorella Using sp. FACHB-9 Chlorella as the culture object, Chlorella in the logarithmic growth phase was inoculated into the culture medium under aseptic conditions and cultured for 4 to 7 days at a temperature of 22℃ to 32℃ with light intensity varying with concentration to obtain high-concentration Chlorella solution. S20. Add the chitosan-modified rope-shaped artificial aquatic plants, which serve as the immobilization carrier, to a high-concentration Chlorella solution and fix it in a constant-temperature shaker at 25℃~35℃ and 120rpm~180rpm for 20h~30h. Then remove it and drain the water to obtain immobilized Chlorella.

2. The method for immobilizing Chlorella according to claim 1, characterized in that, The culture medium is BG11 medium at a concentration of 2.5 to 3.5 times.

3. A method for immobilizing Chlorella according to claim 1 or 2, characterized in that, The BG11 culture medium comprises: 1.50 g sodium nitrate, 0.04 g dipotassium hydrogen phosphate, 0.075 g magnesium sulfate heptahydrate, 0.036 g calcium chloride, 0.020 g sodium carbonate, 0.006 g citric acid, 0.006 g ferric ammonium citrate, 0.001 g disodium EDTA, 2.86 mg boric acid, 1.86 mg manganese chloride tetrahydrate, 0.222 mg zinc sulfate heptahydrate, 0.39 mg sodium molybdate dihydrate, 0.079 mg copper sulfate pentahydrate, 0.049 mg cobalt nitrate hexahydrate, and 1000 mL distilled water.

4. A method for immobilizing Chlorella according to claim 1, 2, or 3, characterized in that, The preparation method of BG11 medium is as follows: mix all components and adjust the pH to 7.1-7.5, and then sterilize at 121℃ for 20 minutes in an autoclave.

5. A method for immobilizing Chlorella according to any one of claims 1 to 4, characterized in that, Under aseptic conditions, inoculate Chlorella salina solution in the logarithmic growth phase into the culture medium at an inoculation rate of 1% to 10%.

6. The method for immobilizing Chlorella according to claim 1, characterized in that, Light intensity: 80 μmol / m 2 / s ~ 150 μmol / m 2 / s.

7. An immobilized Chlorella, characterized in that, Prepared by the method described in any one of claims 1 to 6.

8. An application of immobilized Chlorella prepared by the method according to any one of claims 1 to 6, characterized in that, Immobilized Chlorella can be used to remove nitrogen and phosphorus from aquaculture wastewater.

9. The application according to claim 8, characterized in that, The inoculum concentration for immobilized Chlorella is 1 g / L to 3 g / L.

10. The application according to claim 8, characterized in that, The algal sludge obtained after the aquaculture wastewater treatment is used to develop aquatic feed, fertilizer, bioenergy or health products.

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