Method for purifying rural domestic sewage by using microalgae and co-producing algal liquid fertilizer and application thereof

Abstract

The present application belongs to the field of environmental protection, and particularly relates to a method for purifying rural domestic sewage by using microalgae and co-producing algal liquid fertilizer and application thereof. The method comprises the following steps: pretreating and evaluating the water quality adaptability of the rural domestic sewage to obtain pretreated sewage; inoculating microalgae into the pretreated sewage, adding a five-element trace beneficial element combination, and carrying out first-stage culture; inducing the microalgae after the first-stage culture by four-dimensional stress; harvesting the microalgae to obtain algal slurry, and separately disposing high-salt or high-EC supernatant; pre-enzymolysis of the algal slurry by a composite enzyme, high-pressure homogenization cracking, and solid-liquid separation to obtain algal liquid fertilizer stock solution. The method realizes the coupling of rural domestic sewage purification, nitrogen and phosphorus resource recovery, and algal source fertilizer preparation.

Description

Technical Field

[0001] This invention belongs to the field of environmental protection, specifically relating to a method for purifying rural domestic sewage and producing algal liquid fertilizer using microalgae, and its application. Background Technology

[0002] Rural domestic sewage contains large amounts of nutrients such as nitrogen and phosphorus. Direct discharge will lead to eutrophication of water bodies; however, resource utilization can turn waste into treasure, achieving a win-win situation for pollution control and resource recovery. Traditional rural domestic sewage treatment technologies, such as activated sludge processes and constructed wetlands, can effectively remove nitrogen and phosphorus, but they generally suffer from problems such as difficult sludge disposal, low resource recovery rates, and high operating costs, making it difficult to achieve high-value recovery and utilization of nitrogen and phosphorus nutrients in sewage.

[0003] Microalgae are single-celled organisms capable of autotrophic growth using light energy and CO2, characterized by rapid growth, high photosynthetic efficiency, and strong environmental adaptability. Microalgae can efficiently absorb and utilize nutrients such as nitrogen and phosphorus from wastewater, converting them into microalgal biomass rich in proteins, lipids, and polysaccharides, thus producing valuable biomass resources while purifying wastewater. However, existing microalgae wastewater treatment technologies still face the following core technological bottlenecks in practical applications: First, rural domestic sewage has a complex composition and low microalgae biomass yield. The quality of rural domestic sewage fluctuates greatly due to factors such as region, season, and farmers' water usage habits. It has a low C / N ratio and contains substances that may inhibit microalgae growth. Under cultivation conditions without targeted optimization, microalgae biomass yield is often low, resulting in limited nitrogen and phosphorus removal efficiency and overall poor economic viability.

[0004] Second, the low added value of microalgae biomass products limits its commercialization pathways. Currently, the main uses of microalgae biomass include biodiesel, animal feed, and bioplastics. However, these bulk products have limited added value and weak market competitiveness, making it difficult to support the independent commercial operation of microalgae wastewater treatment technology. Obtaining high-value-added microalgae derivatives is key to promoting the commercialization of microalgae wastewater treatment technology.

[0005] Third, there is insufficient understanding and targeted regulation of biostimulants in microalgae. Studies have shown that microalgal cells naturally contain a variety of biostimulants beneficial to plant growth (…). biostimulantsThese biostimulants include plant hormones (such as indole-3-acetic acid (IAA), cytokinins, and gibberellins), free amino acids, active polysaccharides, betaine, and polyamines. These substances can promote plant growth and improve the quality of agricultural products from multiple dimensions, including promoting photosynthesis, regulating carbon and nitrogen metabolism, enhancing stress resistance, and providing nutrition. However, under conventional microalgae culture conditions, the intracellular content of these biostimulants is not high, and current technologies lack systematic methods for directionally increasing the content of biostimulants in microalgae using culture condition optimization and stress-induced strategies.

[0006] Fourth, it is difficult to simultaneously achieve high cell wall disruption efficiency and high retention rate of active substances in microalgae. Microalgae cell walls are densely structured, with cellulose and pectin as their framework. Conventional direct application of algal solutions makes it difficult for plants to effectively absorb and utilize the active substances within the microalgae cells. Post-treatment techniques such as chemical lysis, enzymatic hydrolysis, or physical cell wall disruption can release intracellular active substances, but existing technologies often face the dilemma of "high cell wall disruption rate leading to low activity retention rate, and high activity retention rate leading to insufficient cell wall disruption rate," lacking a synergistic optimization scheme between cell wall disruption efficiency and active substance retention.

[0007] Therefore, there is an urgent need to develop an integrated technology that combines efficient rural domestic sewage purification, high-yield cultivation of microalgae biomass, targeted enrichment of biostimulants, and preparation of high-efficiency algal liquid fertilizer, so as to realize the transformation from "pollutant removal" to "high-value resource recovery". Summary of the Invention

[0008] In view of the problems existing in the prior art, the purpose of this invention is to design and provide a method for purifying rural domestic sewage and producing algal liquid fertilizer using microalgae, and its application.

[0009] The present invention is specifically implemented using the following technical solutions: The first aspect of this invention provides a method for purifying rural domestic sewage and co-producing algal fertilizer using microalgae, comprising: pretreating and assessing the water quality suitability of rural domestic sewage to obtain pretreated sewage; inoculating microalgae into the pretreated sewage and adding a combination of five beneficial trace elements for a first-stage cultivation; inducing four-dimensional stress on the microalgae after the first-stage cultivation; harvesting the induced microalgae to obtain algal sludge, and separately treating the high-salt or high-EC supernatant produced by stress induction; and performing pre-enzymatic hydrolysis with a compound enzyme and high-pressure homogenization lysis on the algal sludge to obtain algal fertilizer stock solution through solid-liquid separation.

[0010] Furthermore, the water quality compatibility assessment includes the detection of NH4. + -N, EC, pH, CODcr, TN and TP, and based on the test results, the water body is determined to be directly compatible, adjustable compatible or temporarily uncompatible.

[0011] Furthermore, the microalgae are one or more of Chlorella, Scenedesmus, Spirulina or Haematococcus pluvialis.

[0012] Furthermore, the five-element trace element combination includes silicon source, rare earth salt, selenium source, and vitamin B. 12 And biotin.

[0013] Furthermore, in the first stage of cultivation, the concentration of microalgae biomass reached 2.0–2.8 g / L, and the NH4+ concentration reached [missing information]. + After the removal rate of -N is not less than 80% and the removal rate of TP is not less than 75%, the four-dimensional stress induction process begins.

[0014] Furthermore, the four-dimensional stress induction includes nutritional stress, high light stress, salt or osmotic stress, and precursor addition.

[0015] Furthermore, the high-salt or high-EC supernatant is reused, diluted, desalinated, or subjected to independent salt control treatment, and is not directly discharged as irrigation water for farmland.

[0016] Furthermore, the pre-enzymatic hydrolysis of the compound enzyme uses cellulase and pectinase, and the temperature of the algal solution is controlled not to exceed 35°C during the high-pressure homogenization process.

[0017] Furthermore, the algal fertilizer stock solution contains at least three of the following: IAA, cytokinin, GA3, free amino acids, water-soluble polysaccharides, and betaine.

[0018] The second aspect of this invention provides the application of the algal fertilizer stock solution prepared by the above method in foliar spraying, root drip irrigation or fertigation of rice, wheat, tomato or cucumber.

[0019] The present invention has the following beneficial effects: (1) This application uses rural domestic sewage as the nitrogen and phosphorus nutrient source for microalgae cultivation. By establishing a water quality compatibility assessment system, optimizing basic culture conditions, adding a combination of trace beneficial elements, and implementing multi-dimensional stress induction treatment, it achieves high-yield accumulation of microalgae biomass and targeted enrichment of various biostimulants.

[0020] (2) This application uses a combination lysis technology to efficiently release intracellular active substances and prepare algal liquid fertilizer rich in a variety of biostimulants. It is then applied to the cultivation of grain crops and vegetable crops to increase yield and improve quality, thereby achieving significant economic and ecological benefits.

[0021] (3) High wastewater purification efficiency and safer water flow destination. After the first stage of microalgae cultivation, the ammonia nitrogen removal rate is ≥80% and the total phosphorus removal rate is ≥75%. The low-salt supernatant of the first stage can be used as an irrigation water source or reused for water distribution when it meets the "Standards for Irrigation Water Quality" (GB 5084-2021). The high-salt or high-EC supernatant of the second stage is treated separately and is not directly discharged as irrigation water.

[0022] (4) Microalgal biomass production is significantly increased. Through the synergistic effect of the combination of five beneficial trace elements, microalgal biomass production is increased by 40% to 60% compared with conventional BG11 culture conditions, and algal protein content is increased by more than 15%.

[0023] (5) The targeted enrichment of biostimulants is outstanding. Through the four-dimensional stress induction strategy, the content of IAA increased by 4 to 8 times compared with the first stage (up to 5 to 8 mg / g DW), cytokinin increased by 5 to 10 times, gibberellin increased by 3 to 5 times, and betaine increased by 3 to 6 times, realizing the simultaneous and efficient enrichment of multiple biostimulants in microalgal cells.

[0024] (6) The combined lysis process has a high cell wall breakage rate and a high activity retention rate. The total cell wall breakage rate of the combined enzymatic hydrolysis and high pressure homogenization process is ≥90%, and the active substance retention rate is ≥85%, which effectively solves the dilemma of traditional single lysis methods.

[0025] (7) The algal liquid fertilizer product has comprehensive activity and multiple functions. The prepared algal liquid fertilizer contains a variety of plant hormones, free amino acids, active polysaccharides, betaine and mineral nutrients, and has multiple functions such as promoting growth, enhancing stress resistance and providing nutrition. It can be used as a biostimulated fertilizer.

[0026] (8) Significant effects on crop yield and quality improvement. Foliar spraying on rice can increase yield by 10% to 15%, improve head rice rate by 4 to 6 percentage points, and reduce chalkiness by 2 to 4 percentage points; root drip irrigation on greenhouse tomatoes can increase yield by more than 16%, increase soluble solids by 13% to 15%, increase vitamin C by 26% to 33%, increase lycopene by 27% to 34%, and increase root activity by 40% to 53%.

[0027] (9) The process is highly integrated, easy to operate, and low in cost. Raw materials (rural domestic sewage, microalgae strains, and trace minerals) are inexpensive and readily available. The water quality compatibility assessment system greatly simplifies the nutrient preparation process, and in most cases, sewage can be used directly without preparation. The equipment and operation are relatively simple, making it suitable for local promotion and application in rural areas, with a net increase in income of over 2,400 yuan per mu. Attached Figure Description

[0028] Figure 1 This is a process flow diagram of the method of the present invention. Detailed Implementation

[0029] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0030] Example 1: A method for purifying rural domestic sewage and producing algal liquid fertilizer using microalgae S1. Pretreatment and water quality adaptability control of rural domestic sewage.

[0031] S1-1 Collection and Primary Sedimentation. Collect rural domestic sewage (including toilet sewage, kitchen sewage and washing wastewater), first pass it through a screen (5-10 mm gap) to intercept large suspended solids and floating matter, and then pass it through a horizontal flow sedimentation tank (hydraulic retention time 1-2 hours) to remove sand and most of the settleable suspended solids, to obtain the supernatant.

[0032] S1-2 Water Quality Testing and Compatibility Assessment. The pH, conductivity (EC), chemical oxygen demand (CODcr), ammonia nitrogen (NH3-N), total nitrogen (TN), and total phosphorus (TP) concentrations of the supernatant were measured. Based on the three-tiered assessment criteria established in Table 1, it was determined whether the wastewater could be directly used for microalgae cultivation. Table 1. Water Quality Compatibility Assessment Standards for Rural Domestic Sewage Used for Microalgae Cultivation .

[0033] If all indicators are within the "suitable range," proceed directly to step S1-4 disinfection. If they are within the "usable range," make the following simple adjustments before proceeding to S1-4: When ammonia nitrogen > 80 mg / L, dilute with low-concentration grey water at a volume ratio of 1:1 to 1:2; when EC > 2500 μS / cm, mix with low-EC grey water; when pH > 8.0, carefully add dilute hydrochloric acid (0.1 mol / L) to adjust the pH to 7.0–7.5; when CODcr is high, appropriately increase the aeration time or mix with low-COD grey water. If the indicators exceed the "usable range," it is recommended to first perform preliminary anaerobic treatment in a septic tank to reduce ammonia nitrogen and EC before using it for microalgae cultivation.

[0034] S1-3 Nutrient Fine-tuning (only when necessary). Most rural domestic wastewater is within the "suitable" or "usable" range after the S1-2 assessment, and the nutrient concentration is already close to the optimal range required for microalgae growth. Only minor adjustments are needed under the following specific conditions; otherwise, nutrient adjustment is not required: C / N ratio adjustment: Sodium acetate should be added only when the C / N ratio is <5:1 (significant carbon source deficiency) to bring the C / N ratio to 8-10:1; no adjustment is needed when the C / N ratio is 5-12:1, because under commensal conditions, microalgae can utilize both CO2 and organic matter in wastewater as carbon sources.

[0035] N / P ratio adjustment: Only when the N / P ratio is >25:1, supplement with potassium dihydrogen phosphate (KH2PO4) to bring the N / P ratio to 10-15:1; no adjustment is needed when the N / P ratio is between 7:1 and 20:1, as this range is already within the acceptable N / P ratio range for microalgae.

[0036] TP concentration: Only when TP < 2 mg / L, supplement KH2PO4 until TP reaches 3-5 mg / L.

[0037] TN concentration: Generally, no supplementation is needed. The TN in rural domestic sewage (30-50 mg / L) is already within the suitable range for microalgae (40-100 mg / L).

[0038] S1-4 Disinfection. The treated wastewater is then subjected to ultraviolet disinfection (254 nm, dose 40–80 mJ / cm²). 2 Alternatively, in emergency situations, sodium hypochlorite can be used for disinfection (5-10 mg / L of available chlorine, dechlorinated after 30 min of contact) to reduce competition and predation of microalgae by bacteria and protozoa, thus obtaining pretreated wastewater.

[0039] S2. Microalgae inoculation and high-yield cultivation – Phase 1 (biomass accumulation phase) S2-1 Algal Strain Selection and Inoculation. Select microalgae strains that exhibit good adaptability to rural domestic sewage environments, rapid growth rates, and the potential to synthesize biostimulants. The microalgae strains may be selected from Chlorella vulgaris (…). Chlorella sp.), Scenedesmus ( Scenedesmus sp.), Spirulina ( Arthrospira platensis ) or Haematococcus pluvialis ( Haematococcus rainy One or more mixed algal species from the group consisting of Chlorella. Preferably, Chlorella (…) is used. Chlorella vulgaris ) and Scenedesmus ( Oblique scene A mixture of algal species was inoculated at a dry weight ratio of 1:1 to 2:1 to improve the comprehensive utilization capacity of different forms of nitrogen (ammonia nitrogen, nitrate nitrogen, and organic nitrogen) in wastewater. Algal species in the logarithmic growth phase were inoculated at a density of 0.2–0.5 g / L (dry weight) into a culture container or photobioreactor containing the pretreated wastewater obtained in step S1, with a culture depth of 15–30 cm.

[0040] S2-2 Basic Culture Condition Control. The culture conditions are controlled as follows: Lighting conditions: Natural light supplemented by LED artificial light source, with a light intensity of 4000–6000 Lux and a light-dark cycle of 14h:10h–16h:8h. This light range ensures efficient photosynthesis of microalgae while avoiding growth restriction due to excessively low light or photoinhibition due to excessively high light.

[0041] Temperature: Maintain the culture medium temperature at 24–30°C, which is the optimal growth temperature range for most microalgae.

[0042] pH value: By intermittently introducing air containing 1%–5% CO2 (v / v) (aeration rate 0.1–0.3 vvm), the pH value is naturally buffered by the carbonic acid system formed by CO2 dissolving in water, maintaining the pH value at 7.0–8.0. Introducing CO2 has the dual function of stabilizing pH and supplementing inorganic carbon source, which is more economical and environmentally friendly than simply adding acids and alkalis. Stirring: Mechanical stirring or airlift stirring is used at a stirring speed of 50–150 rpm to ensure uniform suspension of microalgae and sufficient gas-liquid exchange.

[0043] S2-3 Addition of a combination of beneficial trace elements. Following inoculation (day 0), a five-element combination of beneficial trace elements, optimized through orthogonal experiments, was added to the culture medium in a single dose. The components, their concentrations, and mechanisms of action are as follows: Table 2. Combination of beneficial trace elements and their effects .

[0044] The synergistic mechanism of the above-mentioned combination of five beneficial trace elements is as follows: silicon can promote the construction of microalgal cell walls and photosynthetic efficiency; rare earth elements (such as lanthanum and cerium) can enhance the photosynthetic phosphorylation activity of microalgae at low concentrations, promoting chlorophyll synthesis and cell division; selenium is a component of glutathione peroxidase, which can improve the antioxidant capacity of microalgae and reduce oxidative damage during cultivation; vitamin B... 12 Biotin and other micro-coenzymes are essential microalgal growth factors, participating in one-carbon metabolism and fatty acid synthesis, respectively. These five components work synergistically across four dimensions: cell structure, photosynthetic energy production, antioxidant protection, and core metabolism. This can increase microalgal biomass yield by 40%–60% and algal protein content by more than 15% compared to conventional BG11 culture medium.

[0045] S2-4 Determination of the first stage of cultivation endpoint. The first stage of cultivation ends when the microalgal biomass concentration reaches 2.0–2.5 g / L (dry weight), and the ammonia nitrogen concentration in the culture medium drops to below 20% of the initial concentration (i.e., ammonia nitrogen removal rate ≥ 80%) and the total phosphorus removal rate ≥ 75%. At this point, the microalgae have completed the efficient absorption of nitrogen and phosphorus from the wastewater, and a large amount of biomass has accumulated, laying a sufficient biomass foundation for the second stage of stress induction.

[0046] S3, Stress-induced – Biostimulant-directed enrichment stage (second stage) After the first stage of cultivation, nitrogen and phosphorus supplementation was stopped, and the cultivation conditions were changed to implement multi-dimensional stress-induced treatment on the microalgae to stimulate their secondary metabolic response and promote the biosynthesis and intracellular accumulation of various plant hormones and other biostimulants. The stress-induced treatment employed a four-dimensional combination strategy of "nutrient stress + high light stress + salt stress + plant hormone precursor addition," lasting for 24–48 hours. S3-1 Nutritional Stress. Stop supplementing with any nitrogen and phosphorus sources, allowing the total nitrogen concentration in the culture medium to naturally decrease to below 5 mg / L and the total phosphorus concentration to decrease to below 1 mg / L, maintaining a nitrogen-phosphorus starvation state. Nitrogen-phosphorus starvation can activate the secondary metabolic switch of microalgal cells, redirecting carbon metabolic flow from primary metabolism (protein and nucleic acid synthesis) to secondary metabolic pathways (shikimic acid pathway, terpene synthesis pathway, etc.), providing the carbon skeleton and energy for plant hormone synthesis.

[0047] S3-2 High Light Stress. The light intensity was gradually increased to 15000–25000 Lux, and the light duration was extended to 20h:4h–24h:0h (continuous light). Under high light conditions, the photosynthetic electron transport chain of microalgae produces a large amount of reactive oxygen species (ROS). Appropriate amounts of ROS, acting as signaling molecules, can activate the expression of genes related to plant hormone synthesis, including the YUCCA family genes in the IAA synthesis pathway and the IPT gene in the cytokinin synthesis pathway, thereby promoting the endogenous synthesis of IAA and cytokinins.

[0048] S3-3 Salt stress or osmotic stress. Add sodium chloride (NaCl) to the culture medium to achieve a salt concentration of 0.1–0.5 mol / L (preferably 0.2 mol / L, approximately 11.7 g / L), and maintain this concentration for 12–48 hours. Salt stress can induce the activity of betaine synthase (BADH) and key enzymes in the polyamine synthesis pathway (such as arginine decarboxylase ADC and ornithine decarboxylase ODC) within microalgal cells, promoting the accumulation of betaine and polyamines (such as putrescine, spermidine, and spermine). Both betaine and polyamines are important biostimulants with osmotic regulation and antioxidant protection functions in plants.

[0049] S3-4 Addition of plant hormone precursors. At the start of stress induction, add one or more of the following plant hormone synthesis precursors to the culture medium: L-Tryptophan (Trp, a direct precursor for IAA synthesis): 50–200 mg / L (preferably 100 mg / L).

[0050] Methionine (Met, a precursor for the synthesis of ethylene and polyamines): 50–150 mg / L.

[0051] Mevaleric acid (MVA, a precursor for the synthesis of terpenoids and cytokinins): 10–50 mg / L.

[0052] L-Tryptophan is a direct precursor to IAA (indole-3-acetic acid) biosynthesis. Microalgal cells can efficiently convert exogenous L-Tryptophan into IAA using tryptophan synthase via tryptophan-dependent pathways (indole-3-pyruvate pathway, IPA pathway). Methionine is a common precursor to the synthesis of ethylene and polyamines (putrescine → spermidine → spermine), promoting the accumulation of spermidine and spermine, and indirectly promoting cytokinin synthesis through the methionine cycle. The exogenous addition of precursors can further directionally promote the synthesis and accumulation of specific biostimulants under stress conditions, achieving a synergistic effect of "opening the synthetic pathway + providing sufficient raw materials".

[0053] The innovation of the four-dimensional stress-induced strategy lies in its approach: it is not simply a matter of superimposing multiple stress treatments, but rather a logically progressive scheme designed based on the microalgal secondary metabolic regulatory network. Nutritional stress initiates secondary metabolic pathways, high light stress provides ROS signals to activate gene expression, salt stress induces the synthesis of specific protective substances, and precursor addition provides the raw materials for synthesis. These four factors exhibit causal logic and synergistic amplification effects, ultimately resulting in a 4-8 fold increase in IAA content, a 5-10 fold increase in cytokinin, a 3-5 fold increase in gibberellin, a 2-5 fold increase in total free amino acids, and a 3-6 fold increase in betaine content compared to the first stage.

[0054] S4. Microalgae harvesting and concentration After the stress induction process is completed, the microalgae are harvested and concentrated. Pre-concentration: The culture medium is pre-concentrated using either natural sedimentation (standing for 6–12 hours) or dissolved air flotation (air-to-water ratio 5%–15%). Dissolved air flotation releases microbubbles to float microalgal cells to the surface, collecting the algal slurry (water content 95%–98%) and the supernatant.

[0055] Further concentration: The pre-concentrated algal slurry is further concentrated by centrifugation (3000-5000 rpm, 10-15 min) or ultrafiltration membrane (molecular weight cutoff 100 kDa) to obtain algal mud (moisture content 80%-90%).

[0056] The nitrogen and phosphorus concentrations in the pre-concentrated supernatant and centrifuged supernatant have been significantly reduced (ammonia nitrogen removal rate ≥80%, total phosphorus removal rate ≥75%). The effluent quality meets the requirements of the "Standard for Irrigation Water Quality" (GB 5084-2021) and can be collected for use as irrigation water for farmland or returned to step S1 for recycling as blending water, thereby reducing water consumption.

[0057] S5. Microalgae lysis and preparation of algal fertilizer The algal sludge obtained in step S4 is subjected to pyrolysis to fully release the various biostimulants accumulated within the microalgal cells. This invention employs a combined pyrolysis process of "enzymatic hydrolysis + high-pressure homogenization," which combines the gentleness of enzymatic hydrolysis with the high efficiency of high-pressure homogenization. Step 1 – Enzymatic Pretreatment: A compound enzyme preparation of cellulase and pectinase is added to the algal sludge. The cellulase activity is ≥10000 U / g, and the pectinase activity is ≥5000 U / g. The total amount of the compound enzyme preparation added is 1%–3% of the dry weight of the algal sludge. Enzymatic hydrolysis is carried out for 4–8 hours at pH 4.5–5.5 and temperature 45–55℃. During the enzymatic hydrolysis, cellulase degrades the cellulose skeleton in the microalgal cell wall, and pectinase degrades the pectin polysaccharides in the cell wall mucilage layer, initially disrupting the cell wall structure and achieving a cell wall disruption rate of 50%–60%. The enzymatic hydrolysis conditions are mild (medium temperature, weakly acidic) and will not cause significant degradation of intracellular active substances, with an activity retention rate of ≥95%.

[0058] Step 2 – High-Pressure Homogenization for Deep Cell Wall Disruption: The enzymatically hydrolyzed algal solution is passed through a high-pressure homogenizer and treated 2–4 times (preferably 3 times) at a pressure of 60–120 MPa (preferably 80 MPa). Under high pressure, the algal solution passes through the narrow gap of the homogenizing valve, experiencing intense shearing, impact, and cavitation effects, resulting in complete disruption of the microalgal cell walls and full release of intracellular substances. After high-pressure homogenization, the total cell wall disruption rate is ≥90%.

[0059] Synergistic advantages of the combined process: Enzymatic hydrolysis initially degrades the cell wall polysaccharide skeleton, reducing the mechanical strength of the cell wall and decreasing the pressure and number of treatments required for subsequent high-pressure homogenization; high-pressure homogenization utilizes physical force to thoroughly break down cells, compensating for the limited degradation efficiency of enzymatic hydrolysis on the cell walls of certain algal species. The entire process is temperature-controllable (enzymatic hydrolysis ≤55℃, homogenization can be cooled), with no strong acids or alkalis used, no organic solvent residue, and a final active substance retention rate ≥85%.

[0060] After lysis, cell debris was removed by centrifugation or filtration, and the clarified algal fertilizer stock solution was collected. This stock solution contains the following active ingredients (calculated based on the combined dry weight of the algae after stress induction and the amount released during lysis, assuming a final yield of 1 L of algal fertilizer stock solution per liter of culture medium): Table 3. Main active ingredients and content range of algae fertilizer stock solution .

[0061] The algae fertilizer concentrate can be concentrated or diluted according to actual needs. Surfactants (such as Tween-80, 0.05% to 0.1% by mass / volume) can be added to enhance leaf adhesion and permeability, and preservatives (such as potassium sorbate, 0.1% by mass / volume) can be added to extend the shelf life, thus producing a finished liquid algae fertilizer product.

[0062] S6. Application of algal liquid fertilizer in crops S6-1 Foliar application on rice. Apply the algae fertilizer solution obtained in step S5 to the rice foliage: Apply during the tillering stage, booting stage (or initial heading stage), and early grain-filling stage, for a total of 2-3 applications, with an interval of 10-15 days between each application. Before application, dilute the algae fertilizer solution with water 100-500 times (preferably 300 times), applying 30-50 L / mu (preferably 40 L / mu) of diluted solution each time. Spraying should be done on sunny mornings before 9:00 AM or after 4:00 PM, avoiding periods of high temperature and strong sunlight, ensuring the leaves are evenly moistened on both sides. Spraying can be done using a backpack sprayer or a plant protection drone.

[0063] S6-2 Application of root drip irrigation for dryland vegetables. Apply the algae fertilizer obtained in step S5 to the root drip irrigation of greenhouse vegetables (such as tomatoes, cucumbers, peppers, etc.): application periods are seedling stage, flowering stage, fruit enlargement stage, and peak fruiting stage, for a total of 3-5 drip irrigations (preferably 4 times) throughout the entire growth period, with an interval of 15-20 days between each application. Before application, dilute the algae fertilizer concentrate with water 200-400 times (preferably 300 times), and apply it evenly to the crop roots through the drip irrigation system with the irrigation water. The amount of diluted solution used each time is 2-5 L / plant (preferably 3 L / plant). During operation, first drip clean water for 5-10 minutes to moisten the soil, then drip the diluted algae fertilizer, and finally drip clean water for 5-10 minutes to flush the pipes to prevent algae fertilizer residue from clogging the drippers.

[0064] Example 2: Using Chlorella to treat rural domestic sewage and produce algal liquid fertilizer for application as foliar spraying on rice. 1. Wastewater pretreatment and water quality adaptation Wastewater from the equalization tank of a rural domestic sewage treatment plant in Zhejiang Province was collected. After treatment by a screen (8 mm gap) and a sedimentation tank (HRT=1.5 h), the supernatant was collected. Measured water quality: pH 7.3, EC 1820 μS / cm, CODcr 265 mg / L, ammonia nitrogen 62 mg / L, TN 48 mg / L, TP 4.2 mg / L.

[0065] The following assessments were conducted based on the compatibility evaluation criteria in Table 1: Ammonia nitrogen 62 mg / L → within the “suitable range” (20–80 mg / L).

[0066] EC 1820 μS / cm → within the “suitable range” (500~2500 μS / cm).

[0067] pH 7.3 → within the "suitable range" (7.0~8.0).

[0068] CODcr 265 mg / L → within the “suitable range” (100~300 mg / L).

[0069] All four indicators were within the "suitable range". No nutrient salts were needed. The medium was directly sterilized with ultraviolet light (254nm, dose 60 mJ / cm²) and then used as a culture medium for later use.

[0070] 2. First Stage – High-Yield Microalgae Cultivation Chlorella ( Chlorella vulgaris The algal species (FACHB-8) was used. In a 200 L flat-plate photobioreactor, 150 L of the pretreated wastewater was added as the culture medium, and the algal species in the logarithmic growth phase was inoculated at an initial inoculation density of 0.3 g / L (dry weight).

[0071] Culture condition control: Lighting: Natural light supplemented by LED lights, with an intensity of 5000 Lux and a light-dark cycle of 16h:8h.

[0072] Temperature: 26±2℃.

[0073] pH: Intermittently introduce compressed air containing 3% CO2 (airflow rate 0.2 vvm) to maintain pH at 7.0–7.8.

[0074] Stirring: Mechanical stirring, 100 rpm.

[0075] The following trace element combination was added once after inoculation (based on culture medium volume): sodium silicate 30 mg / L (as SiO2), lanthanum chloride (LaCl3·7H2O) 1.0 mg / L, sodium selenate (Na2SeO4) 0.3 mg / L (as Se), and vitamin B12. 12 1.0 μg / L, Biotin 0.8 μg / L.

[0076] Daily sampling and monitoring of microalgal biomass concentration (OD measurement) 680 (And converted to dry weight) and the concentrations of ammonia nitrogen and total phosphorus in the culture medium. After 9 days of cultivation, the concentration of microalgae biomass reached 2.3 g / L (dry weight). At this time, the concentration of ammonia nitrogen in the culture medium had decreased from the initial 62 mg / L to 7.5 mg / L (removal rate 87.9%), and the concentration of total phosphorus had decreased from 4.2 mg / L to 0.8 mg / L (removal rate 81.0%), thus ending the first stage of cultivation.

[0077] The basic biostimulant content of algal samples in the first stage was determined as follows: IAA 1.9 mg / g DW, zeatin (cytokinin) 0.30 mg / g DW, gibberellin GA3 1.1 mg / g DW, total free amino acids accounted for 31% of the dry weight of algae, and betaine accounted for 1.3% of the dry weight of algae.

[0078] 3. Second stage – Stress-induced enrichment of biostimulants Stop adding any nitrogen and phosphorus sources, change the culture conditions, and implement four-dimensional stress induction treatment: Nutritional stress: Allow TN in the culture medium to drop naturally to below 5 mg / L and TP to drop to below 1 mg / L.

[0079] High light stress: The light intensity was gradually increased from 5000 Lux to 20000 Lux, and the light cycle was adjusted from 16h:8h to continuous illumination (24h:0h).

[0080] Salt stress: Add NaCl to the culture medium to a final concentration of 0.2 mol / L.

[0081] Precursor addition: Add L-tryptophan 100 mg / L simultaneously.

[0082] The stress-induced treatment lasted for 48 hours. At the end, the microalgal biomass concentration was approximately 2.5 g / L (dry weight). Samples were taken to determine changes in the content of various biostimulants in the algae. Table 4 Comparison of intracellular biostimulant content in microalgae before and after stress induction .

[0083] 4. Microalgae harvesting and lysis for preparation of algal liquid fertilizer Pre-concentration was carried out using dissolved air flotation (air-to-water ratio 10%) to obtain algal slurry with a water content of approximately 96%. The algal slurry was then centrifuged at 4000 rpm for 12 minutes to obtain algal mud with a water content of 84%.

[0084] The combined lysis process of "enzymatic hydrolysis + high-pressure homogenization" was adopted: Step 1: Enzymatic hydrolysis: Add a complex enzyme preparation of cellulase and pectinase to the algal mud at 2% of the dry weight of the algal mud, adjust the pH to 5.0, and enzymatically hydrolyze for 6 hours at a constant temperature of 50℃.

[0085] The second step is high-pressure homogenization: the enzymatically hydrolyzed algal solution is passed through a high-pressure homogenizer and treated three times at a pressure of 80 MPa.

[0086] After lysis, the cells were removed by centrifugation (8000 rpm, 15 min), and the clear algal fertilizer stock solution was collected.

[0087] The measured concentrations of active ingredients in the algae fertilizer stock solution were as follows: IAA 17.2 mg / L, zeatin 3.6 mg / L, GA 37.2 mg / L, free amino acids 1020 mg / L, water-soluble polysaccharides 720 mg / L, and betaine 96 mg / L.

[0088] Add Tween-80 (0.08%, v / v) and potassium sorbate (0.1%, w / v) to the stock solution, stir well, and then fill into a container to produce a finished liquid algae fertilizer.

[0089] 5. Field trial of foliar spraying on rice A field experiment was conducted in a paddy field in Yiqiao Town, Xiaoshan District, Hangzhou City. The rice variety used was "Yongyou 1540," and the soil type was paddy soil. The experiment included three treatments, each with three replicates, and each plot was 30 m². 2 Randomized block permutation: Control (CK): Conventional fertilization (basal fertilizer + tillering fertilizer + panicle fertilizer), with equal amounts of water sprayed at the tillering stage, heading stage and early grain filling stage.

[0090] Algae liquid fertilizer treatment (T1): The conventional fertilization is the same as CK. The algae liquid fertilizer stock solution is diluted with water 300 times and sprayed on the leaves at the tillering stage, the heading stage and the early grain filling stage, with a spraying amount of 40 L / mu each time.

[0091] Commercially available amino acid fertilizer treatment (T2): Conventional fertilization is the same as CK. Commercially available amino acid foliar fertilizer (main components are amino acids ≥100 g / L and trace elements ≥20 g / L) is sprayed according to the dosage recommended in the product instructions. The spraying time and number of times are the same as T1.

[0092] Spraying was carried out on sunny mornings between 8:00 and 9:00 AM using a backpack electric sprayer, ensuring the rice leaves were evenly moistened on both sides. Yield was assessed at harvest, and rice quality indicators were measured. The results are shown in Table 5. Table 5. Effects of different treatments on rice yield and rice quality .

[0093] Note: Different lowercase letters in the same row indicate significant differences between treatments. P <0.05), the same applies below.

[0094] The results showed that the rice yield treated with the algae-based liquid fertilizer of this invention reached 598 kg / mu, an increase of 14.6% compared with the water control and 9.7% compared with commercially available amino acid foliar fertilizer. In terms of rice quality, the head rice rate of the algae-based liquid fertilizer treatment increased by 5.4 percentage points and the chalkiness decreased by 3.4 percentage points compared with the control, and the appearance and processing quality of the rice were significantly improved.

[0095] 6. Wastewater purification effect After the first stage of cultivation, the water quality of the low-salt supernatant was as follows: ammonia nitrogen 7.5 mg / L (removal rate 87.9%), total phosphorus 0.8 mg / L (removal rate 81.0%), CODcr 82 mg / L (removal rate 69.1%), and pH 7.5. This low-salt supernatant, meeting the "Standards for Irrigation Water Quality" (GB 5084-2021), can be used as an irrigation water source for surrounding farmland. The high-salt or high-EC supernatant produced by the second stage of salt stress or osmotic stress is collected and treated separately and is not directly used for irrigation.

[0096] Comparative Example 1: No added trace beneficial elements Same as Example 2, but without adding the five-element trace beneficial element combination in steps S2-3, otherwise completely identical.

[0097] Results: In the first stage, the concentration of microalgal biomass was only 1.4 g / L (2.3 g / L in Example 1, a decrease of 39.1%). After stress induction, the IAA content in the algae was 4.2 mg / g DW (7.5 mg / g DW in Example 1, a decrease of 44.0%). Ultimately, the yield increase of rice fertilized with algal extract was 7.8% (14.6% in Example 1). This indicates that the combination of beneficial trace elements not only promotes the accumulation of microalgal biomass but also significantly contributes to subsequent IAA enrichment and the final yield increase.

[0098] Comparative Example 2: Stress-induced without the addition of precursors Same as Example 2, but L-tryptophan is not added in steps S3-4, only nutritional stress + high light stress + salt stress are applied.

[0099] Results: The IAA content in the algae after stress induction was 4.8 mg / g DW (7.5 mg / g DW in Example 1, a decrease of 36.0%), indicating that the addition of exogenous precursors contributed about 36% to the directed synthesis of IAA.

[0100] Comparative Example 3: Single Fracturing Method Same as Example 2, but in step S5 only high-pressure homogenization (80 MPa, 3 times) was used, without enzymatic pretreatment. Results: The total cell wall breakage rate was 73% (92% in Example 1 combined process), and the IAA concentration in the algal fertilizer stock solution was 11.3 mg / L (17.2 mg / L in Example 1, a decrease of 34.3%), indicating that the combined lysis process has a significantly better efficiency in releasing active substances than the single method.

[0101] Table 6 Summary of comparisons between each comparative example and Example 1 .

[0102] Example 3: Mixed algae treatment mode: Chlorella ( Chlorella vulgaris) and Scenedesmus ( Oblique scene The mixed algal species were mixed at a dry weight ratio of 1:1, with a total inoculation density of 0.4 g / L (based on dry weight). The remaining steps and conditions were the same as in Example 2.

[0103] After 8 days of cultivation in the first stage, the concentration of microalgae biomass reached 2.6 g / L (13.0% higher than that of Chlorella monoculture in Example 1), with an ammonia nitrogen removal rate of 91.5% and a total phosphorus removal rate of 88.2%. The mixed algae species have a stronger comprehensive utilization capacity for different forms of nitrogen (ammonia nitrogen, nitrate nitrogen, and a small amount of organic nitrogen) in wastewater.

[0104] After stress induction, the IAA content in the algae was 8.1 mg / g DW (an increase of 8.0% compared to Example 1), and the IAA concentration in the stock solution of the algae fertilizer was 18.6 mg / L. When the algae fertilizer was foliar sprayed on rice using the same method as in Example 1, the yield increase reached 16.2%.

[0105] The results showed that the mixed algal species mode was superior to the Chlorella monoculture mode in terms of purification efficiency, biostimulant yield, and crop yield increase.

[0106] Example 4: Application of algae liquid fertilizer in drip irrigation of greenhouse tomatoes 1. Basic Information about the Experimental Site The experiment was conducted at a facility vegetable base in Xiaoshan District, Hangzhou City, Zhejiang Province. The test crop was autumn-winter tomato (variety "Zhefen 202"), cultivated in a solar greenhouse. Basic soil physicochemical properties: pH 6.8, EC 580 μS / cm, organic matter 18.5 g / kg, available nitrogen 85 mg / kg, available phosphorus 32 mg / kg, available potassium 145 mg / kg. Tomatoes were transplanted on September 5, 2024, with a plant spacing of 35 cm and a row spacing of 60 cm, irrigated and fertilized using a drip irrigation system.

[0107] 2. Preparation of Algal Fertilizer The algal fertilizer stock solution was prepared according to steps S1 to S5 of Example 1. The concentration of active ingredients in the stock solution was the same as in Example 1 (IAA 17.2 mg / L, zeatin 3.6 mg / L, GA3 7.2 mg / L, free amino acids 1020 mg / L, water-soluble polysaccharides 720 mg / L, betaine 96 mg / L).

[0108] 3. Experimental Design Four treatments were set up, with each treatment repeated three times. The cell area was 20 m². 2 : Table 7 .

[0109] The algae-based fertilizer was applied once each during the seedling stage (15 days after transplanting), flowering stage (first flower spike opening), fruit enlargement stage (first fruit spike diameter 3-5 cm), and peak fruiting stage (second to third fruit spike enlargement). Root irrigation was conducted via a drip irrigation system. First, clean water was dripped for 10 minutes to moisten the soil, then diluted algae-based fertilizer was applied with the irrigation water, and finally, clean water was dripped for another 10 minutes to flush the pipes. The control treatment received the same amount of clean water via drip irrigation at the same time.

[0110] The foliar spray for the T3 treatment was applied once during the flowering period and once during the fruit enlargement period, ensuring that both sides of the leaves were evenly moistened, and was applied after 16:00.

[0111] 4. Test Results Table 8 Effects of different treatments on tomato yield and quality .

[0112] 5. Results Analysis Treatment T2 (300-fold dilution of algal fertilizer concentrate for root irrigation) showed the best overall benefits. This treatment, in addition to conventional fertilization, applied the algal fertilizer to the tomato roots via a drip irrigation system, with four root irrigations throughout the growing season, achieving the following results: Significant yield increase: The yield per mu reached 6036 kg, an increase of 16.4% compared with the control. The IAA contained in the algae liquid fertilizer promoted the growth of tomato roots and the germination of lateral roots, the cytokinin delayed the senescence of functional leaves and extended the photosynthetic effective period, and the gibberellin promoted the cell division and enlargement of the fruit.

[0113] Quality improvement: Soluble solids increased by 13.5%, vitamin C content increased by 26.3%, lycopene content increased by 27.5%, sugar-acid ratio increased from 12.4 to 15.1, and fruit flavor and nutritional quality were significantly improved.

[0114] Root system promotion: Root fresh weight increased by 29.1% compared to the control, and root activity increased by 44.4%. The well-developed root system enhances the plant's ability to absorb water and nutrients, laying a physiological foundation for high yield and quality in the above-ground parts.

[0115] The yield and quality of T2 and T1 were not significantly different, indicating that a 300-fold dilution of the algal fertilizer had already achieved a relatively good effect, and further increasing the dosage (200-fold dilution) did not yield significant marginal benefits. Although the T3 treatment (low-volume root irrigation + foliar spray) was slightly lower than T1 and T2, it was still significantly better than the control.

[0116] 6. Comparison of foliar spraying for rice and root irrigation for tomatoes Table 9 Comparison of the effects of different application methods of algal liquid fertilizer .

[0117] The above comparison shows that the algal liquid fertilizer prepared by this invention is suitable for both foliar spraying of rice and drip irrigation of greenhouse vegetables. It exhibits stable yield-increasing and quality-improving effects under both different crop types and application methods, demonstrating good broad applicability and promising prospects for widespread application.

Claims

1. A method for purifying rural domestic sewage and co-producing algal liquid fertilizer using microalgae, characterized in that, include: Pre-treatment and water quality suitability assessment of rural domestic sewage were carried out to obtain pre-treated sewage; Microalgae were inoculated into the pretreated wastewater, and a combination of five beneficial trace elements was added for the first stage of cultivation. The microalgae after the first stage of cultivation were subjected to four-dimensional stress induction; the induced microalgae were harvested to obtain algal mud, and the high-salt or high-EC supernatant produced by stress induction was treated separately; the algal mud was subjected to pre-enzymatic hydrolysis with compound enzymes and high-pressure homogenization lysis, and solid-liquid separation was performed to obtain algal fertilizer stock solution.

2. The method according to claim 1, characterized in that, The water quality compatibility assessment includes the detection of NH4. + -N, EC, pH, CODcr, TN and TP, and based on the test results, the water body is determined to be directly compatible, adjustable compatible or temporarily uncompatible.

3. The method according to claim 1, characterized in that, The microalgae are one or more of Chlorella, Scenedesmus, Spirulina or Haematococcus pluvialis.

4. The method according to claim 1, characterized in that, The five-element trace element combination includes silicon source, rare earth salt, selenium source, and vitamin B. 12 And biotin.

5. The method according to claim 2, characterized in that, The first stage of cultivation involves cultivating microalgae until the biomass concentration reaches 2.0–2.8 g / L and the NH4+ concentration reaches 100 g / L. + After the removal rate of -N is not less than 80% and the removal rate of TP is not less than 75%, the four-dimensional stress induction process begins.

6. The method according to claim 1, characterized in that, The four-dimensional stress induction includes nutritional stress, high light stress, salt or osmotic stress, and precursor addition.

7. The method according to claim 1, characterized in that, The high-salt or high-EC supernatant is reused, diluted, desalinated, or treated with independent salt control, and is not directly discharged as irrigation water for farmland.

8. The method according to claim 1, characterized in that, The pre-enzymatic hydrolysis of the compound enzyme uses cellulase and pectinase, and the temperature of the algal solution is controlled not to exceed 35°C during high-pressure homogenization.

9. The method according to claim 1, characterized in that, The algal fertilizer stock solution contains at least three of the following: IAA, cytokinin, GA3, free amino acids, water-soluble polysaccharides, and betaine.

10. The application of the algal fertilizer stock solution prepared by the method according to any one of claims 1 to 9 in the foliar spraying, root drip irrigation or fertigation of rice, wheat, tomato or cucumber.

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