Glass noodles wastewater treatment process based on microalgae

Abstract

The present invention relates to a glass noodles wastewater treatment process based on microalgae, belonging to the technical field of microbial wastewater treatment. Chlorella sorokiniana NZ1 and Monoraphidium sp. NZ4 are selected. An analysis of experiment results shows that when Chlorella sorokiniana NZ1 and Monoraphidium sp. NZ4 are subjected to mixed culturing, the biomass of both in the wastewater reaches the highest level. At the same time, the removal rates of water quality indicators also exhibit the most significant improvement; specifically, the removal rates of COD, TN, ammonia nitrogen, and TP can respectively reach 83.4%, 91.19%, 81.51%, and 78.71%. Thus, it can be concluded that subjecting the algal strains NZ1 and NZ4 to mixed culturing is the most effective glass noodles wastewater treatment method.

Description

A microalgae-based vermicelli wastewater treatment process Technical Field

[0001] The invention relates to a microalgae-based vermicelli wastewater treatment process, belonging to the technical field of microbial wastewater treatment. Background Art

[0002] Currently, my country has a large number of vermicelli production enterprises using miscellaneous beans as raw materials. However, although the wastewater generated during the vermicelli production process is non-toxic, it is somewhat harmful. For every ton of vermicelli produced, 12-15 tons of high-concentration organic wastewater is generated. This organic wastewater is rich in various nutrients such as soluble protein, starch, dietary fiber, and oligosaccharides, and also has extremely high levels of COD and SS (suspended solids concentration). Vermicelli wastewater is discharged in large quantities and at high concentrations. If discharged directly without treatment, it will cause the water to turn black and stink, causing environmental degradation and even contaminating groundwater sources. Therefore, as the vermicelli industry flourishes, corresponding wastewater treatment problems have also arisen. The treatment of this type of wastewater faces problems such as high processing difficulty, high investment costs, and severe waste of resources. There is an urgent need to find a green, economical, environmentally friendly, and sustainable method for treating vermicelli wastewater.

[0003] Chinese patent document CN103508621A (application number 201210219902.5) discloses a system for treating vermicelli production wastewater. After anaerobic reaction, the wastewater achieves a COD removal rate of 90%. Chinese patent document CN110550832A (application number 201910989225.7) discloses a system and method for recycling corn starch wastewater. After treatment, the wastewater achieves complete recovery of water, carbon, nitrogen, and phosphorus resources. The system efficiently recovers water resources, with a water reuse rate exceeding 55%; efficiently recovers carbon resources, with a methane recovery rate of up to 85%; and efficiently recovers nitrogen and phosphorus resources, with nitrogen recovery rates exceeding 85% and phosphorus recovery rates exceeding 82%. Chinese patent document CN114014505A (application number 202111458549.1) discloses a method for treating corn starch processing wastewater using microalgae. After the wastewater is treated with Chlorella HQ, the chemical oxygen demand removal rate can reach 91.28%; the total phosphorus removal rate can reach 78.33%; and the total nitrogen removal rate and ammonia nitrogen removal rate reach 65.51% and 67.33% respectively.

[0004] Vermicelli wastewater is rich in soluble protein, starch, dietary fiber, oligosaccharides and other nutrients. If these substances are utilized while treating the wastewater, certain economic benefits will be generated. At present, there are not many studies on the treatment of vermicelli wastewater and its conversion into economically valuable by-products. The output substances are relatively single, mostly protein products. Chinese patent document CN110305831A (application number 201910619486.X) discloses a method for producing fishery growth hormone protein using vermicelli wastewater and its application. The bacterial liquid after fermentation of vermicelli wastewater can be mixed with feed and fed to fish, or it can be directly added to the feed during feed processing and fed to fish. It has a significant growth-promoting effect on fish growth and a significant production-increasing effect. Chinese patent document CN105859357A (application number 201610289845.6) discloses a method for preparing microbial fertilizer using by-products generated during vermicelli production. This patent utilizes the by-products generated during vermicelli production, treating the wastewater as a microbial seed culture medium, and using the powder residue and black powder as carriers for microbial fertilizer. Through microbial fermentation, the valuable components such as starch, protein, and amino acids in the by-products are fully utilized to achieve comprehensive resource utilization. Chinese patent document CN101302062A (application number 200810138006.X) discloses a low-pollution, biophilic treatment process for wastewater from vermicelli production. After the vermicelli wastewater is treated, the protein in the starch production wastewater is extracted using an acid precipitation method, a composite coagulant rapid intensification method, and a protein ripening recovery unit ripening recovery method, and the protein is used to make protein feed.

[0005] Currently, the treatment of vermicelli wastewater faces challenges such as high difficulty, high investment costs, and complex equipment. Chinese patent document CN101302062A (application number 200810138006.X) discloses a low-pollution biophilic treatment process for wastewater from vermicelli production. After passing through a screen and sedimentation process, the vermicelli wastewater must enter an anaerobic tank with an upflow anaerobic sludge bed, an aerobic tank equipped with an aerobic bioreactor, a biofilter, an oxidation pond, a sand filter tank, and other treatment processes. Chinese patent document CN103508621A (application number 201210219902.5) discloses a treatment system for vermicelli production wastewater. The wastewater passes through a first sedimentation tank, an acidification regulating tank, a water lift pump, an anaerobic reactor, a second sedimentation tank, and an aerobic treatment tank before it can meet discharge standards. These treatment methods require extensive equipment and high investment costs. Therefore, it is important to find a green, environmentally friendly, and sustainable method for treating vermicelli wastewater.

[0006] Microalgae are single-celled microorganisms that can grow in a variety of aquatic environments. Their short growth cycles and high efficiency in utilizing light energy have made them highly sought after. Microalgae can utilize carbon dioxide and nutrients (such as nitrogen and phosphorus) in water for growth, making them a promising approach for wastewater treatment. They can even thrive in many harsh water quality environments, such as wastewater, brackish water, and even highly saline waters. Compared to traditional wastewater treatment methods, microalgae can significantly improve the removal efficiency of nutrients, organic pollutants, heavy metals, and pathogens from wastewater, and their ability to tolerate toxic substances makes them suitable for a wide range of treatment applications. Furthermore, microalgae are rich in biomass, including carbohydrates, proteins, and lipids. Their metabolites can be used to produce a variety of products, including animal feed, medical supplies, food, and renewable energy. Microalgae also have the ability to absorb CO2 and can accumulate biomass using nutrients such as nitrogen and phosphorus from various wastewaters, converting them into proteins, fatty acids, and other cellular components. Research has shown that some microalgae can contain oil, exceeding 50% of their dry weight.

[0007] Microalgae are rich in fatty acids, of which linoleic acid and α-linolenic acid are two very important components. Both play an indispensable role in human and animal health, but are often lacking in the daily diet and are in urgent need of supplementation. In particular, linoleic acid has multiple benefits, including lowering blood lipids, softening blood vessels, lowering blood pressure, and promoting microcirculation. It can effectively prevent or reduce the incidence of cardiovascular disease. It is particularly beneficial for the prevention and treatment of diseases such as hypertension, hyperlipidemia, angina pectoris, coronary heart disease, atherosclerosis, and senile obesity. It can effectively prevent the deposition of human serum cholesterol in blood vessel walls, and is therefore known as a "vascular scavenger." It has significant health benefits in preventing and treating atherosclerosis and cardiovascular disease. Although the production of fatty acids from microalgae has been proven to be feasible, there are still relatively few patents related to the production of essential fatty acids and oils from microalgae.

[0008] At present, microalgae have achieved certain results in the treatment of different wastewaters, such as industrial wastewater, municipal wastewater, agricultural wastewater and livestock breeding wastewater, but research in food processing wastewater and other aspects still needs to be strengthened. Such as the screening of microalgae strains, optimization of culture conditions, etc. In the future, further in-depth research can be combined with modern biotechnology to promote the application of microalgae in the field of vermicelli wastewater treatment, so as to achieve efficient utilization of resources and sustainable development of the environment. According to the characteristics of vermicelli wastewater, suitable microalgae strains are selected for cultivation, which can effectively reduce the concentration of nutrients in the wastewater, improve water quality, and reduce pressure on the environment. At the same time, rich biomass can also be produced during the growth process of microalgae, which has certain resource utilization value. The present invention mainly studies the use of microalgae to treat vermicelli processing wastewater, cultivates microalgae with vermicelli wastewater, not only harmlessly treats the vermicelli wastewater, but also utilizes the biomass in the microalgae for resource utilization, realizing the combination of vermicelli wastewater treatment process and production process.

[0009] Summary of the Invention

[0010] To address the shortcomings of existing technologies, the present invention provides a microalgae-based process for treating vermicelli wastewater. Two target algae strains, Chlorella sorokiniana NZ1 and Monoraphidium sp. NZ4, were screened and obtained. Compared to single-strain cultures, the combined culture of these two strains exhibited higher treatment efficiency and greater adaptability to vermicelli wastewater, enabling more effective nutrient removal from the wastewater.

[0011] The technical solutions of the present invention are as follows:

[0012] A microalgae-based vermicelli wastewater treatment process comprises the following steps:

[0013] (1) Chlorella NZ1 and Monotrichia NZ4 were cultured in BG-11 liquid medium to obtain Chlorella NZ1 seed solution and Monotrichia NZ4 seed solution;

[0014] (2) The seed liquid of Chlorella NZ1 and the seed liquid of Monochamus NZ4 in step (1) were combined and inoculated into the vermicelli wastewater. The initial inoculation amount was OD 680 =0.1-0.2, light cultivation treatment of vermicelli wastewater.

[0015] Preferably, according to the present invention, the Chlorella NZ1 described in step (1) was deposited in the China Center for Type Culture Collection (CCTCC) on March 13, 2023, and the deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is: CCTCC NO: M 2023313.

[0016] Preferably, according to the present invention, the monochaeta NZ4 described in step (1) was deposited in the China Center for Type Culture Collection (CCTCC) on October 20, 2023, and the deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is: CCTCC NO: M 20231949.

[0017] According to the present invention, preferably, in step (1), the culture is expanded to the logarithmic growth phase.

[0018] According to the preferred embodiment of the present invention, the conditions for the expanded culture in step (1) are: a temperature of 25-30°C, a light intensity of 40-45 μmol photons·m -2 ·s -1 Next cultivation.

[0019] According to the preferred embodiment of the present invention, the components per liter of the BG-11 liquid culture medium in step (1) are as follows:

[0020] 10 mL of 100×BG-11 solution, 1 mL of 6 mg / mL ammonium ferric citrate solution, 1 mL of 20 mg / mL Na2CO3 solution, 1 mL of 30.5 mg / mL K2HPO4 solution, and the balance water;

[0021] The components per liter of 100×BG-11 solution are as follows:

[0022] NaNO3 149.6 g, MgSO4·7H2O 7.5 g, CaCl2·2H2O 3.6 g, citric acid 0.6 g, pH 8.0, 0.25 M Na2EDTA 1.12 mL, trace elements 100 mL; the trace element composition per liter is as follows: H3BO3 2.86 g, ZnSO4·7H2O 0.22 g, MnCl2·4H2O 1.81 g, Na2MoO4·2H2O 0.39 g, CuSO4·5H2O 0.079 g, Co(NO3)2·6H2O 0.049 g.

[0023] According to the present invention, preferably, the volume ratio of the Chlorella NZ1 seed solution and the Monoacanthus NZ4 seed solution when combined in step (2) is 1:(0.5-3); more preferably 1:1.

[0024] According to the preferred embodiment of the present invention, the initial inoculum size in step (2) is OD 680 =0.1.

[0025] According to the preferred embodiment of the present invention, the conditions for light culture in step (2) are: culture temperature of 25-30°C, light intensity of 40-45 μmol photons·m -2 ·s-1 , the culture time is 10-15 days; further preferably, the culture time is 12 days.

[0026] Preferably, according to the present invention, the water quality indicators of the vermicelli wastewater in step (2) are as follows: chemical oxygen demand is 15800 mg / L, ammonia nitrogen content is 128.82 mg / L, total nitrogen content is 270.48 mg / L, total phosphorus content is 105.90 mg / L, and pH value is 3.3-3.8.

[0027] Chlorella sorokiniana NZ1 was deposited in the China Center for Type Culture Collection (CCTCC) on March 13, 2023. The deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is: CCTCC NO: M 2023313.

[0028] Monoraphidium sp. NZ4 was deposited in the China Center for Type Culture Collection (CCTCC) on October 20, 2023. The deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is: CCTCC NO: M 20231949. Beneficial effects:

[0029] 1. The present invention screened out Chlorella sorokiniana NZ1 and Monoraphidium sp. NZ4. Experimental analysis showed that when Chlorella sorokiniana NZ1 and Monoraphidium sp. were co-cultured, their biomass in wastewater reached the highest level. Furthermore, the removal rates of water quality indicators were significantly improved. Specifically, the removal rates for COD, TN, ammonia nitrogen, and TP reached as high as 83.4%, 91.19%, 81.51%, and 78.71%, respectively. Therefore, it can be concluded that the co-culture of algae strains NZ1 and NZ4 is the most effective method for treating vermicelli wastewater.

[0030] 2. Mixed cultivation of algae strains NZ1 and NZ4 was carried out in vermicelli wastewater, and the OD 680 , chlorophyll a content and carotenoid content all reached the highest level. Specifically, at the end of the culture period, OD 680 The content of chlorophyll a and carotenoids reached 25.91 mg / L and 8.44 mg / L, respectively. In addition, the mixed culture of algae strains NZ1 and NZ4 also achieved the maximum oil yield, reaching 256.79 mg / L.

[0031] 3. The mixed culture of Chlorella sorokiniana NZ1 and Monoraphidium sp. NZ4 of the present invention can be used to treat vermicelli wastewater and simultaneously produce oil. Combining the production of Chlorella sorokiniana NZ1 and Monoraphidium sp. NZ4 biomass with the purification of vermicelli wastewater lays a foundation for the clean treatment and resource utilization of vermicelli wastewater, while also providing a strategy for the large-scale production of microalgae oils. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is a microscopic image of algae strain NZ1 and algae strain NZ4.

[0033] Figure 2 is a phylogenetic tree of algal strains NZ1 and NZ4 constructed based on 18S rDNA sequences.

[0034] Figure 3 shows the removal of wastewater quality indicators of original vermicelli wastewater under different ventilation rates.

[0035] Figure 4 shows the growth of different algae strain combinations in vermicelli wastewater.

[0036] Figure 5 shows the change curves of chlorophyll a content (A) and carotenoid content (B) when different algae strain combinations were cultured in vermicelli wastewater.

[0037] Figure 6 is a bar graph of oil production and oil content when different algae strain combinations were cultured in vermicelli wastewater.

[0038] Figure 7 shows the COD change curve (A) and the COD removal rate column (B) of the wastewater when different algae strain combinations are cultured in vermicelli wastewater.

[0039] FIG8 is a curve showing the change of ammonia nitrogen in wastewater (A) and a bar graph showing the removal rate of ammonia nitrogen in wastewater (B) when different algae strain combinations are cultured in vermicelli wastewater.

[0040] Figure 9 is a curve showing the change of total nitrogen in wastewater (A) and a bar graph showing the removal rate of total nitrogen in wastewater (B) when different algae strain combinations are cultured in vermicelli wastewater.

[0041] Figure 10 is a curve showing the change of total phosphorus in wastewater (A) and a bar graph showing the removal rate of total phosphorus in wastewater (B) when different algae strain combinations are cultured in vermicelli wastewater. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto.

[0043] The sources of experimental materials used in the examples are as follows:

[0044] Algae strains NZ1 and NZ4 were isolated and purified from vermicelli wastewater and wastewater treatment sludge in Zhaoyuan, Shandong Province. Algae strain SP1 was purchased from Shanghai Guangyu Biotechnology Co., Ltd. with the product number (GY-D19 Chlorella sp.).

[0045] Vermicelli wastewater, sourced from pea vermicelli processing at a vermicelli factory in Zhaoyuan, Yantai City, Shandong Province, is used for microalgae cultivation. The water quality indicators are as follows: chemical oxygen demand (COD) of 15,800 mg / L, ammonia nitrogen content of 128.82 mg / L, total nitrogen (TN) content of 270.48 mg / L, total phosphorus (TP) content of 105.90 mg / L, and a pH of 3.3-3.8.

[0046] Corn starch wastewater, sourced from a starch processing plant in Shandong Province, is used for microalgae cultivation. The water quality indicators for this wastewater are as follows: chemical oxygen demand (COD) of 9093.33 mg / L, ammonia nitrogen content of 45.81 mg / L, total nitrogen (TN) content of 757.69 mg / L, total phosphorus (TP) content of 18.30 mg / L, and a pH range of 3.5-4.5.

[0047] The culture medium composition involved in the embodiment is as follows:

[0048] The components of BG-11 liquid medium per liter are as follows:

[0049] 10 mL of 100×BG-11 solution (without Fe, phosphate, or carbonate), 1 mL of 6 mg / mL ammonium ferric citrate solution, 1 mL of 20 mg / mL Na2CO3 solution, 1 mL of 30.5 mg / mL K2HPO4 solution, and the balance water.

[0050] BG-11 solid medium is prepared by adding the following components per liter to BG-11 liquid medium:

[0051] 3 g of sodium thiosulfate, 1 mol / L NaOH solution adjusted to pH 8.2, 10 mL of 1 mol / L tris(hydroxymethyl)methylaminoethanesulfonic acid solution, and 15 g of agar powder.

[0052] The components per liter of 100×BG-11 (excluding Fe, phosphate, and carbonate) are as follows:

[0053] NaNO3 149.6 g, MgSO4·7H2O 7.5 g, CaCl2·2H2O 3.6 g, citric acid 0.6 g, Na2EDTA (pH 8.0, 0.25 M) 1.12 mL, trace elements 100 mL; the trace element composition per liter is as follows: H3BO3 2.86 g, ZnSO4·7H2O 0.22 g, MnCl2·4H2O 1.81 g, Na2MoO4·2H2O 0.39 g, CuSO4·5H2O 0.079 g, Co(NO3)2·6H2O 0.049 g;

[0054] Ammonium ferric citrate solution (6 mg / mL, 1000×): Weigh 0.6 g of ammonium ferric citrate, dissolve it in 100 mL of ddH2O, and store it in a refrigerator at 4°C until use.

[0055] Na2CO3 solution (20 mg / mL, 1000×): Weigh 2 g of Na2CO3 and dissolve it in 100 mL of ddH2O. Store in a refrigerator at 4°C until use.

[0056] K2HPO4 solution (30.5 mg / mL, 1000×): Weigh 3.05 g of K2HPO4 and dissolve it in 100 mL of ddH2O. Store in a refrigerator at 4°C until use.

[0057] 1 mol / L tris(hydroxymethyl)aminoethanesulfonic acid solution: Weigh 114.62 g of tris(hydroxymethyl)aminoethanesulfonic acid (TES) and dissolve it in 500 mL of ddH2O. Adjust the pH to 8.2 with 1 mol / L NaOH solution and store in a refrigerator at 4°C until use.

[0058] The expansion culture of algae strains was carried out in BG-11 medium.

[0059] Example 1: Screening and identification of algae strains NZ1 and NZ4

[0060] Microalgae were separated from vermicelli wastewater and wastewater treatment sludge samples from Zhaoyuan, Shandong Province using plate separation technology and cultured on BG-11 solid culture medium plates. The specific steps are as follows:

[0061] The sludge sample was initially diluted with sterile water and filtered with gauze to remove plankton and larger suspended particles. The diluted suspension was then graded diluted with sterile water to 10 -1 , 10 -2 , 10 -3 , 10 -4 0.5 mL of dilution of each concentration was added to the surface of BG-11 solid culture medium, spread evenly with a sterile applicator, and then placed upside down at a temperature of 30°C and a light intensity of 40 μmol photons·m -2 ·s-1 Cultivate in a light-incubator. Subsequently, using the plate separation technique, single algal colonies of varying morphology, size, and color are streaked onto BG-11 solid medium for purification. Repeat this process at least 3-5 times until a single algal species is purified. Inoculate a single algal colony into a 100mL Erlenmeyer flask containing 50mL of BG-11 liquid medium and incubate on a shaker under the same culture conditions to accelerate microalgae enrichment. After enrichment, examine the microalgae for morphology and appearance under a microscope to confirm purification. Also, apply the algal liquid to LB solid medium to verify the growth of contaminants and confirm the completion of purification.

[0062] Two algal species were initially purified based on colony size, morphology, and color, designated strains NZ1 and NZ4. Cultures grown in BG-11 liquid medium were then observed microscopically and initially differentiated based on morphology, color, and size (Figure 1). Genomic DNA from the microalgae was extracted using a modified CTAB method. 18S rDNA sequences were amplified by PCR using the 18S rDNA primers 18S-F: 5'-CCTGGTTGATCCTGCCAGTAG-3' and 18S-R: 5'-TTGATCCTTCTGCAGGTTCA-3'. The PCR products were purified by 1% agarose gel electrophoresis, cloned by TA, and sent to Qingdao Qingke Biotechnology Co., Ltd. for sequencing. The resulting 18S rDNA sequences for strain NZ1 are shown in SEQ ID NO. 1, and those for strain NZ4 are shown in SEQ ID NO. 2. The sequencing results were compared using GenBank Blast and analyzed using MEGA 4.0 to construct a phylogenetic tree, as shown in Figure 2 .

[0063] The taxonomic status of the isolated and purified algae was determined through comprehensive analysis of their colony characteristics, cell morphology, and 18S rDNA sequencing results. Microscopic observation revealed that strain NZ1, based on its morphological characteristics, was a spherical, single-celled algae measuring 5-10 μm in size, dark green in color, with distinct pigmentation, visible fission, and lacking flagella, similar in morphology to Chlorella vulgaris. Strain NZ4 was an ellipsoidal, single-celled algae with a larger end and smaller end, measuring 2-8 μm in size and lacking flagella, similar in morphology to Chlorella vulgaris. Phylogenetic comparisons revealed that strain NZ1 was closely related to Chlorella sorokiniana, while strain NZ4 was closely related to Monoraphidium sp.

[0064] Chlorella sorokiniana NZ1 was deposited in China Center for Type Culture Collection (CCTCC) on March 13, 2023. The deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. The deposit number is: CCTCC NO: M 2023313;

[0065] Monoraphidium sp. NZ4 was deposited in the China Center for Type Culture Collection (CCTCC) on October 20, 2023. The deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is: CCTCC NO: M 20231949.

[0066] Example 2: Effects of different ventilation rates on water quality indicators of vermicelli wastewater

[0067] The study of the effects of different ventilation volumes on the water quality indicators of vermicelli wastewater was conducted in the following steps:

[0068] (1) The vermicelli wastewater was subjected to solid-liquid separation and filtered through 8 layers of gauze to separate fine solids;

[0069] (2) 1.5 L of the wastewater from step (1) was placed in a 3 L conical flask and aerated using an electromagnetic air compressor pump, with the flow rate controlled by a glass rotor flowmeter. Two aeration rates were set: 1 L / min and 2 L / min. A non-aerated control group was also established. Samples were taken every 24 hours and water quality indicators such as COD, total nitrogen, ammonia nitrogen, and total phosphorus were measured.

[0070] The experimental results are shown in Figure 3. Under aerated culture conditions, water quality indicators in vermicelli wastewater declined significantly faster than under static culture (no aeration), with the difference being highly significant. This difference is likely due to the fact that, under static culture (no aeration), gas exchange occurs solely at the liquid surface, resulting in a relatively slow absorption of nutrients by microorganisms in the vermicelli wastewater. Aeration, on the other hand, enhances the transfer of nutrients between the microorganisms and the wastewater, significantly increasing the gas exchange rate and thus promoting microbial growth. Different aeration rates also showed significant differences in the removal of water quality indicators. Under aeration of 2 L / min, the rate of decline in water quality indicators was higher than under aeration of 1 L / min. This may be because low aeration prevented sufficient contact between the microorganisms and air, thereby impairing the absorption process and, in turn, the reduction in water quality indicators. Furthermore, the most significant reduction in water quality indicators occurred after three days of aeration. However, on the fourth day of aeration, a slight upward trend in water quality indicators (ammonia nitrogen) was observed, possibly due to poor microbial growth and the subsequent death of some microorganisms. In summary, without inoculating exogenous microorganisms, the optimal aeration rate of vermicelli wastewater is 2 L / min, and the aeration lasts for 3 days.

[0071] Example 3: Effects of mixed culture of algae strains in vermicelli wastewater on microalgae and wastewater

[0072] A microalgae-based vermicelli wastewater treatment process comprises the following steps:

[0073] (1) Chlorella vulgaris NZ1, Chlorella vulgaris NZ4, and Chlorella vulgaris SP1 were cultured in BG-11 liquid medium to the logarithmic growth phase to obtain microalgae seed liquid;

[0074] (2) The microalgae seed liquid of step (1) was combined and inoculated into vermicelli wastewater, the pH value was natural, and the initial inoculation amount was OD 680 =0.1, the combination of algae species is shown in Table 1, and the vermicelli wastewater is treated by light culture. The culture temperature is 30 °C and the light intensity is 45 μmol photons·m -2 ·s -1 , ventilation volume is 2L / min.

[0075] Table 1. Combination of algae species

[0076] 1. Impact on microalgae growth

[0077] Samples were taken every 48 hours and OD was measured 680, the growth curves of microalgae cells in single culture and mixed culture modes are shown in Figure 4. The results showed that the growth curves of microalgae showed an "S" shape regardless of whether it was a single algae species or a mixed algae species culture, indicating that the three microalgae were able to effectively utilize the nutrients in the wastewater to achieve normal growth. In the mixed algae culture mode, the biomass yield was significantly higher than that of a single algae strain culture. Specifically, the mixed culture of algae strains NZ1 and NZ4 showed the most outstanding biomass yield and was the optimal combination. Under these conditions, the OD 680 The value reached 6.664, which was higher than the OD value when NZ1, NZ4 and SP1 were cultured alone. 680 The values ​​increased by 44.00%, 66.93% and 54.50% respectively. This shows that the mixed cultivation of microalgae in vermicelli wastewater can significantly increase the biomass yield of microalgae.

[0078] 2. Effect on chlorophyll a content in microalgae

[0079] Every 48 hours, algae liquid was collected to determine the chlorophyll a content. An improved chlorophyll a determination method was used: 2 mL of algae liquid was placed in a 2 mL centrifuge tube and centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded and 2 mL of methanol was added and pipetted to mix. Vortexed for 5 minutes in the dark, the mixture was allowed to stand for 10 minutes, and then centrifuged again at 10,000 rpm for 10 minutes. The supernatant was collected for determination, using methanol as a reference. The absorbance was measured at wavelengths of 666 nm and 653 nm using a UV spectrophotometer. The chlorophyll a content was determined according to the following formula. 叶 =15.65×OD 666 -7.34×OD 653

[0080] Where:

[0081] C 叶 is the chlorophyll a concentration, mg / L;

[0082] OD λ is the absorbance value at wavelength λ.

[0083] The data in Figure 5A show that the chlorophyll a content of the algae strains showed an overall upward trend throughout the cultivation process. Chlorophyll a increased rapidly during the initial growth phase, eventually reaching a plateau. After 12 days of cultivation, the chlorophyll a contents of strains NZ1, NZ4, and SP1 were 9.07, 1.83, and 19.64 mg / L, respectively. Under monoculture conditions, strain SP1 had the highest chlorophyll a content, reaching 19.64 mg / L, while NZ4 had the lowest, at only 1.83 mg / L. Under mixed culture conditions, the highest chlorophyll a content was achieved in a mixed culture of NZ1 and NZ4, reaching a plateau of 25.91 mg / L on day 12. However, mixed cultures of other algae species did not reach the final chlorophyll a content achieved when strains NZ1 and NZ4 were mixed. This may be due to the lack of competition from other algae when cultured alone, but in polyculture, other algae share the same nutrient resources, which can complicate resource allocation and hinder SP1 from maintaining its highest chlorophyll content. Furthermore, the mixed culture of strains NZ1 and NZ4 may alter their growth and metabolic patterns, favoring chlorophyll a accumulation.

[0084] 3. Effect on carotenoid content in microalgae

[0085] The carotenoid content was determined every 48 hours. The carotenoid content was determined as follows: 2 mL of algal solution was centrifuged at 13,000 rpm for 10 min, the precipitate was resuspended in 1 mL of N,N-dimethylformamide solution, pipetted to mix, and centrifuged at 13,000 rpm for 10 min. The supernatant was measured using a spectrophotometer for OD 461 and OD 664 The carotenoid content was calculated according to the following formula. 类 =(OD 461 -0.046×OD 664 )×4

[0086] Where:

[0087] C 类 is the carotenoid concentration, mg / L;

[0088] OD λ is the absorbance value at wavelength λ.

[0089] As shown in Figure 5B, the carotenoid content of the strains showed an overall upward trend. Carotenoid content increased rapidly during the initial growth phase, eventually stabilizing. Under mixed culture conditions, strains NZ1 and NZ4 had the highest carotenoid content, reaching a plateau on day 12, with a carotenoid content of 8.44 mg / L. In contrast, when cultured individually, strains NZ1, NZ4, and SP1 showed similar growth trends in carotenoid content, all reaching a plateau on day 12, with final carotenoid contents of 3.95, 0.90, and 6.47 mg / L, respectively. However, under mixed cultures (strains NZ1 and SP1, and strains NZ4 and SP1), the carotenoid contents remained within the expected range and were lower than the final carotenoid content of strains NZ1 and NZ4. In summary, Figure 5B clearly shows that the carotenoid production reached the highest level when the algal strains NZ1 and NZ4 were mixed cultured, which indicates that the mixed culture of algal strains NZ1 and NZ4 significantly promoted the carotenoid content. It also shows that more carotenoids will be produced when the algal strains NZ1 and NZ4 are mixed to treat wastewater.

[0090] 4. Impact on microalgae oil production

[0091] After 12 days of culture, the algal slurry was collected and its lipid content was determined. A modified chloroform-methanol method was used to determine lipid content: 30 mL of algal slurry was placed in a 50 mL centrifuge tube (volume V). The tube was placed in a centrifuge and centrifuged at 7,000 rpm for 10 minutes. After centrifugation, the supernatant was quickly discarded to obtain an algal slurry containing lipids. 500 μL of 1 mol / L hydrochloric acid solution was added to the slurry and the microalgae were vortexed to disrupt the microalgae. Subsequently, a chloroform / methanol extraction solution (V:V = 2:1) was added to the slurry in a fume hood for lipid extraction. The tube was placed in a shaker protected from light and shaken at 150 rpm for 4 hours to ensure thorough mixing. After centrifugation, 10 mL of normal saline was added to the tube, vortexed for 60 seconds, and then centrifuged again at 7,000 rpm for 10 minutes. After centrifugation, separate the layers. Use a syringe to extract the bottom layer of the chloroform / methanol and oil mixture into a dried tin foil container (tin foil dry weight M0) in a fume hood. Place the tin foil container in the fume hood until all the chloroform and methanol evaporate. Then, place the tin foil container in a 60°C oven and dry to a constant weight, which is then weighed as M1. The oil yield is calculated using the following formula:

[0092] Where: η is the oil content, %; C is the oil yield, g / L; M0 is the mass of the tin foil after drying, mg; M1 is the total mass of the tin foil and oil after drying, mg; V is the volume of the algae solution, mL; M is the dry weight of microalgae, g / L.

[0093] Figure 6 shows the oil production of different algal species combinations in wastewater. As shown in Figure 6, the mixed culture of strains NZ1 and NZ4 had the highest oil content, reaching 43.21%, with an oil yield of 256.79 mg / L. While the oil content of other algal species combinations increased during cultivation in wastewater, these combinations were all lower than the oil yield and oil content of the mixed culture of strains NZ1 and NZ4. When a single algal strain was cultivated in wastewater, the oil yield was the lowest. In summary, the mixed culture of strains NZ1 and NZ4 was the most effective for oil production.

[0094] 5. Analysis of fatty acid composition of microalgae

[0095] The fatty acid composition of the microalgae was analyzed using GC-MS. 50 mL of the algal culture solution from a 12-day mixed culture of strains NZ1 and NZ4 was centrifuged at 7500 × g for 10 minutes. The supernatant was discarded and the microalgae culture was harvested. The microalgae culture was stored at -80°C and then freeze-dried in a vacuum freeze dryer for 12 hours.

[0096] 0.1 g of freeze-dried microalgae culture powder was placed in a centrifuge tube. 10% of the powder's mass of solid acid was added, followed by 2 mL of methanol. The mixture was incubated at 60°C for 40 minutes. Then, 2 mL of a 4% KOH-methanol solution was added. The mixture was placed in a constant-temperature waterbath at 60°C for 1 hour and then cooled. 1 mL of n-hexane was added to the reaction solution, and after ultrasonication for 10 minutes, 200 μL of the supernatant was extracted and placed in a clean reagent bottle. 25 μL of the internal standard (2 mg / mL, methyl heptadecanoate) was then added to the bottle for analysis. Fatty acids were determined using a gas chromatography-mass spectrometer in full-scan mode with a scan range of 50-300 amu. The parameters were as follows: column: VF-23ms, temperature program: 150°C for 1 minute, then ramped to 165°C at 1°C / min; injection port temperature: 220°C.

[0097] The results are shown in Table 2. The content of C16-C18 fatty acids is more than 96.74%, and it contains almost no long-chain hydrocarbon components. The saturated fatty acids (C16:0, C18:0) obtained in the present invention account for 38.46%, which is a relatively low content; the polyunsaturated fatty acids (C18:2, C18:3) account for up to 43.01%, which is much higher than the European standard limit of 12%. Therefore, mixed cultivation of microalgae NZ1 and NZ4 in wastewater is not suitable for use as biodiesel. However, under this condition, the contents of linoleic acid and linolenic acid are 27.80% and 15.21% respectively. Therefore, it is of positive significance to use vermicelli wastewater to cultivate microalgae NZ1 and NZ4 to produce linoleic acid and linolenic acid.

[0098] Table 2. Fatty acid composition of microalgae

[0099] 6. Impact on the removal of pollutants in wastewater

[0100] During the cultivation process, samples were taken every two days, with 20 mL of sample taken each time. The samples were placed in a 50 mL centrifuge tube and centrifuged at 7,000 r / min for 10 minutes. The supernatant was filtered through a 0.45 μm pore size filter membrane to remove the microalgae cells. The filtrate was used to determine the sewage quality indicators. The determination method was based on the "Water and Wastewater Monitoring Method" (China National Standard Monitoring Method). The TN content was determined by potassium persulfate digestion UV spectrophotometry, and the NH4 + -N content was determined by Nessler's reagent spectrophotometry, TP content was determined by potassium persulfate digestion ammonium molybdate colorimetry, and COD was determined by potassium dichromate method. + -Calculation formula for N, TP and COD removal rate: RR=(C0-Ct) / C0

[0101] Where, RR: removal rate (%); C0: initial concentration (mg / L); C t : concentration at time t (mg / L).

[0102] 1) Removal of COD in wastewater

[0103] Figure 7 clearly illustrates the COD removal performance of different algal species combinations in vermicelli wastewater. All algal species combinations demonstrated some COD removal effectiveness, but among these combinations, the mixed culture of strains NZ1 and NZ4 significantly outperformed the other species combinations. Within the first eight days, the mixed culture of strains NZ1 and NZ4 effectively removed a significant amount of COD, ultimately reducing the COD concentration from 15,800 mg / L to 2,622.5 mg / L, a removal rate of 83.4%. In contrast, the other mixed cultures performed relatively poorly in terms of COD removal. Individual cultures of strains NZ1, NZ4, and SP1 showed similar COD removal performance, with final COD concentrations dropping to 3,860 mg / L, 3,847.5 mg / L, and 3,297.5 mg / L, respectively. Furthermore, at the end of the treatment period, COD levels remained relatively stable, likely due to the death of the microalgae due to nutrient depletion, resulting in minimal COD concentration changes in the wastewater.

[0104] 2) NH4 in wastewater + -N removal

[0105] Figure 8 shows that ammonia nitrogen removal was consistently achieved when strains NZ1 and NZ4 were co-cultured in wastewater, ultimately reaching a removal rate of 81.51%, the highest among all experimental groups. The ammonia nitrogen removal rate when individual strains were cultured in wastewater was slower than when combined, stabilizing after the 10th day. Ultimately, the ammonia nitrogen removal rates for strains NZ1, NZ4, and SP1 alone were 68.08%, 33.15%, and 67.99%, respectively. With the exception of the co-culture of NZ1 and NZ4, the ammonia nitrogen removal rates for all other co-cultured strains were within the expected range. In summary, the co-culture of strains NZ1 and NZ4 achieved the best ammonia nitrogen removal results for vermicelli wastewater.

[0106] 3) Removal of TN from wastewater

[0107] Figure 9 shows the total nitrogen removal performance of different algae species combinations in vermicelli wastewater. The results show that all three strains have a certain ability to remove total nitrogen from vermicelli wastewater, with the total nitrogen content showing an overall downward trend. A mixed culture of strains NZ1 and NZ4 reduced the total nitrogen concentration from 270.477 mg / L to 23.82 mg / L, achieving a removal rate of 91.19%, the highest total nitrogen removal rate, exceeding the total nitrogen removal rates of strains NZ1 and NZ4 alone. In summary, a mixed culture of strains NZ1 and NZ4 is the most effective for total nitrogen removal from vermicelli wastewater.

[0108] 4) Removal of TP from wastewater

[0109] Figure 10 shows the total phosphorus removal performance of different algae species combinations in wastewater. It can be seen that the mixed culture of strains NZ1 and NZ4 has the ability to remove total phosphorus from vermicelli wastewater, with an overall downward trend. TP concentrations decreased rapidly over the first 10 days, and by day 12, the total phosphorus removal rate reached 78.71%, the highest, exceeding the total phosphorus removal rates of strains NZ1 and NZ4 alone. The mixed culture of the two strains in wastewater maintained stable total phosphorus removal over the 12 days of treatment, with total phosphorus concentrations decreasing from 105.90 mg / L to 22.54 mg / L. Starting on day 10, treatment efficiency gradually declined, likely due to insufficient nutrients in the wastewater, which restricted microalgae growth and slowed the decline in phosphorus content. In summary, the mixed culture of strains NZ1 and NZ4 was the most effective for total phosphorus removal from vermicelli wastewater.

[0110] Comparative Example 1: Treatment of corn starch wastewater by combined cultivation of algae strains NZ1 and NZ4

[0111] A microalgae-based corn starch wastewater treatment process comprises the following steps:

[0112] (1) Chlorella vulgaris NZ1 and Monochamus punctatus NZ4 were cultured in BG-11 liquid medium to the logarithmic growth phase to obtain microalgae seed liquid;

[0113] (2) The microalgae seed liquid of step (1) was mixed in a volume ratio of 1:1 and inoculated into corn starch wastewater. The pH value was natural and the initial inoculum size was OD 680 =0.1, the starch wastewater was treated by light culture, the culture temperature was 30℃, and the light intensity was 45μmol photons·m -2 ·s -1 , the culture time is 12 days.

[0114] Determine the OD of the microalgae culture starting point 680 and wastewater COD, total phosphorus, total nitrogen, and ammonia nitrogen. The results are shown in Table 3.

[0115] Table 3. Corn starch wastewater water quality indicators

[0116] According to the data in Table 3, the algae strains NZ1 and NZ4 were mixed and cultured in corn starch wastewater. After 12 days of growth, the OD 680 The value only reached 1.776, which is the OD value of the growth in vermicelli wastewater. 680 The values ​​were 26.65%. This indicates that the two algal strains did not grow well in corn starch wastewater and were relatively more suitable for growth in vermicelli wastewater. In terms of water quality indicator removal rates, the removal efficiency in corn starch wastewater was far lower than that in vermicelli wastewater, with COD, total phosphorus, total nitrogen, and ammonia nitrogen removal rates of only 51.35%, 37.52%, 24.09%, and 29.91%, respectively. This suggests that the two algal strains were limited in their ability to treat corn starch wastewater and exhibited significantly greater water quality indicator removal efficiency in vermicelli wastewater. Based on these results, we speculate that this may be due to the higher abundance of nutrients in vermicelli wastewater, such as nitrogen and phosphorus, which are key elements for microbial growth and reproduction, contributing to the enhanced growth rate of microalgae. Furthermore, environmental conditions may be more suitable for the growth of strains NZ1 and NZ4. Vermicelli wastewater may contain chemicals that are more friendly to strains NZ1 and NZ4, or it may contain fewer inhibitory factors compared to corn starch wastewater. In summary, vermicelli wastewater may have characteristics that are more suitable for the growth of algae strains NZ1 and NZ4 than corn starch wastewater, making strains NZ1 and NZ4 more effective in treating vermicelli wastewater. However, the specific influencing factors may require further experimentation and analysis to clarify.

[0117] The present invention separates and purifies microalgae from vermicelli wastewater and wastewater treatment sludge in Zhaoyuan, Shandong Province, and obtains algae strains NZ1 and NZ4. Using these two microalgae to treat vermicelli wastewater, it is found that when algae strains NZ1 and NZ4 are mixed and cultured, they can have the highest biomass accumulation in vermicelli wastewater. The OD value at the end of culture is 0.04. 680The content of chlorophyll a can reach 25.91 mg / L, and the content of carotenoids can reach 8.44 mg / L. The oil production reached the maximum value (256.79 mg / L) when the algae strains NZ1 and NZ4 were mixed and cultured. At the same time, the wastewater quality was well purified, and the removal rates of COD, total nitrogen, ammonia nitrogen, and total phosphorus reached 83.4%, 91.19%, 81.51%, and 78.71%, respectively. Compared with other reports, the treatment effect is better, the treatment equipment is simple and low-cost, and it can produce derivatives such as oil, linoleic acid, linolenic acid, chlorophyll, and carotenoids, with broad application prospects. Microalgae can also be reused to treat wastewater.

[0118] Based on the above results, we speculate that Chlorella NZ1 and Monochamus NZ4 have complementary advantages in vermicelli wastewater treatment. They may have their own advantages in treating different types of pollutants, and through combined cultivation, they can exert synergistic effects on each other and improve wastewater treatment efficiency. Chlorella NZ1 and Monotrichia NZ4 may have strong adaptability to the specific environmental conditions in vermicelli wastewater, allowing them to maintain high growth rates and biological activity in this wastewater. The metabolites of Chlorella NZ1 and Monotrichia NZ4 may promote each other, resulting in a synergistic effect in combined cultivation, improving the treatment capacity of wastewater pollutants. The combination of different algal strains may lead to higher resource allocation efficiency, such as nutrient utilization and biomass accumulation, thereby improving the overall efficiency of wastewater treatment. Chlorella NZ1 and Monotrichia NZ4 may have strong stress resistance and can maintain a good growth state under relatively high-concentration wastewater conditions, thereby effectively treating wastewater. Compared with single-strain microalgae cultivation, mixed cultivation can form a mutually reinforcing and complementary ecosystem by integrating the characteristics and advantages of different algae strains, thereby improving the overall efficiency of wastewater treatment and biomass production. This makes the treatment of vermicelli wastewater more effective than other algae strain treatment methods.

[0119] This invention lays the foundation for further research into optimizing microalgae treatment conditions for vermicelli wastewater, providing insights into increasing microalgae yield and reducing cultivation costs in wastewater. The above-described embodiments provide a detailed description of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific examples of the present invention and are not intended to limit the present invention. Any modifications or improvements made within the scope of the principles of the present invention are intended to be included within the scope of the present invention.

Claims

1. Chlorella sorokiniana NZ1 was deposited in the China Center for Type Culture Collection on March 13, 2023. The deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is: CCTCC NO: M 2023313.

2. Monoraphidium sp. NZ4 was deposited in the China Center for Type Culture Collection on October 20, 2023. The deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is: CCTCC NO: M 20231949.

3. A vermicelli wastewater treatment process based on microalgae, characterized in that: The steps include: (1) Chlorella NZ1 and Monotrichia NZ4 were cultured in BG-11 liquid culture medium to obtain Chlorella NZ1 seed solution and Monotrichia NZ4 seed solution; (2) The seed solution of Chlorella NZ1 and the seed solution of Monochamus NZ4 in step (1) were combined and inoculated into vermicelli wastewater, with an initial inoculation amount of OD 680 =0.1-0.2, light cultivation to treat vermicelli wastewater.

4. The vermicelli wastewater treatment process according to claim 3, characterized in that: The Chlorella NZ1 described in step (1) was deposited in the China Center for Type Culture Collection on March 13, 2023, with the deposit address being: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number being: CCTCC NO: M 2023313; The mononema NZ4 was deposited in the China Center for Type Culture Collection on October 20, 2023. The deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is: CCTCC NO: M 20231949.

5. The vermicelli wastewater treatment process according to claim 3, characterized in that: In step (1), the culture is expanded to the logarithmic growth phase.

6. The vermicelli wastewater treatment process according to claim 3, characterized in that: The conditions for the expanded culture in step (1) are: a temperature of 25-30°C and a light intensity of 40-45 μmol photons·m -2 ·s -1 Next cultivation.

7. The vermicelli wastewater treatment process according to claim 3, characterized in that: The components per liter of the BG-11 liquid culture medium in step (1) are as follows: 100×BG-11 solution 10mL, 6mg / mL ammonium ferric citrate solution 1mL, 20mg / mL Na2CO3 solution 1mL, 30.5mg / mL K2HPO4 solution 1mL, balance water; The components per liter of 100×BG-11 solution are as follows: NaNO3 149.6g, MgSO4·7H2O 7.5g, CaCl2·2H2O 3.6g, citric acid 0.6g, pH 8.0, 0.25M Na2EDTA 1.12mL, trace elements 100mL; the components per liter of the trace elements are as follows: H3BO3 2.86g, ZnSO4·7H2O 0.22g, MnCl2·4H2O 1.81g, Na2MoO4·2H2O 0.39g, CuSO4·5H2O 0.079g, Co(NO3)2·6H2O 0.049g.

8. The vermicelli wastewater treatment process according to claim 3, characterized in that: In step (2), the volume ratio of the Chlorella NZ1 seed solution and the Monoacanthus NZ4 seed solution when combined is 1:(0.5-3); more preferably 1:1; Preferably, the initial inoculum size in step (2) is OD 680 =0.

1.

9. The vermicelli wastewater treatment process according to claim 3, characterized in that: The conditions for the light culture in step (2) are: the culture temperature is 25-30°C, the light intensity is 40-45 μmol photons·m -2 ·s -1 The culture time is 10-15 days; further preferably, the culture time is 12 days.

10. The vermicelli wastewater treatment process according to claim 3, characterized in that: The water quality indicators of the vermicelli wastewater described in step (2) are as follows: chemical oxygen demand is 15800 mg / L, ammonia nitrogen content is 128.82 mg / L, total nitrogen content is 270.48 mg / L, total phosphorus content is 105.90 mg / L, and pH value is 3.3-3.8.

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