A method for treating organic wastewater by using improved microalgae and coupling carbon sequestration

Abstract

The application provides a method for treating organic wastewater by using improved microalgae and coupling carbon fixation, which comprises the following steps: culturing microalgae under high-temperature and high-illumination conditions, improving the gene of cytochrome P450 enzyme of the microalgae by using a gene editing technology, and up-regulating the expression amount of the cytochrome P450 enzyme; gradiently domesticating and culturing the improved microalgae in organic wastewater, inoculating the domesticated microalgae into pretreated organic wastewater, and culturing in a photobioreactor, wherein the organic matters in the wastewater are degraded during the culturing process. The microalgae is improved from the gene level, the expression amount of the cytochrome P450 enzyme is strengthened by stimulating and regulating the high-temperature and high-illumination culturing conditions, the hydroxylation of the organic pollutants is promoted, the organic pollutants are more easily degraded, and carbon dioxide can be fixed. The method can treat various types of organic wastewater, has high treatment efficiency, and has low cost.

Description

Technical Field

[0001] This invention belongs to the field of organic wastewater treatment technology, and relates to a method for treating organic wastewater and coupling carbon fixation using modified microalgae. Background Technology

[0002] With social development and human production activities, pollution problems are inevitable. To avoid wastewater pollution, wastewater treatment is necessary before discharge. Organic wastewater, due to its recalcitrant nature, is one of the most challenging aspects of wastewater treatment. Its sources include numerous production sectors such as chemical, pharmaceutical, fuel, food, and power industries. Besides carbon, organic wastewater often contains nitrogen and phosphorus, which are significant factors contributing to eutrophication. Therefore, the treatment of organic pollutants in wastewater typically requires degrading them to generate harmless small-molecule substances and, as much as possible, converting and utilizing the pollutants.

[0003] Taking a biomass power plant as an example, the combustion of biomass generates steam for power generation. This steam is then cooled by water in a cooling tower. To reduce scaling and corrosion of the coils in the cooling tower, scale and corrosion inhibitors, typically organic compounds, are added to the circulating water. Benzotriazole is one of the active ingredients. To maintain the quality of the circulating water in the cooling tower, wastewater needs to be discharged and replenished periodically. This wastewater usually contains inorganic salts, humic substances, and organic pollutants. Organic pollutants can easily cause toxicity to aquatic organisms and generate scale during the desalination process, affecting system performance and lifespan. Therefore, the wastewater from the cooling tower needs to be treated, especially the treatment of organic pollutants.

[0004] The treatment of organic pollutants can be broadly categorized into physical, chemical, and biological methods. Physical methods, including microfiltration, ultrafiltration, coagulation, and adsorption, often suffer from low removal efficiency and long processing times. Chemical methods, such as traditional precipitation and oxidation, are insufficient for treating organic matter, so electrochemical oxidation can be used, but it is costly and difficult to treat large volumes of wastewater. Biological methods have advantages such as being eco-friendly, resource-recoverable, energy-efficient, and low-cost, and have broad application prospects.

[0005] Microalgae refer to algal communities whose morphology can only be discerned under a microscope. They have a good effect on treating organic wastewater, fixing carbon dioxide and accumulating nutrients such as proteins and lipids through photosynthesis. CN 105621783A discloses a method for removing nitrogen and phosphorus from wastewater using a sequencing batch reactor (SBR). The method includes: pretreatment of nitrogen- and phosphorus-rich wastewater; inoculating the SBR with microalgae; introducing the pretreated wastewater into the reactor; controlling the volumetric exchange rate according to the total nitrogen content in the wastewater; turning on an external light source; aerating the reaction; allowing the algal solution to settle after the reaction; discharging the supernatant; repeatedly introducing wastewater for treatment as needed; and periodically discharging algal sludge. In this method, microalgae replace activated sludge as the main reactant to remove nitrogen and phosphorus from the wastewater. However, the nitrogen and phosphorus in the wastewater exist in the form of inorganic salts, consumed as nutrients for photosynthesis, rather than degrading nitrogen-containing organic matter.

[0006] CN 109368801A discloses a method for treating organic pollutants in water based on a microalgae-assisted Fenton system. The method includes: inoculating microalgae into wastewater, adjusting the pH, setting the temperature, light intensity, and light duration, introducing air mixed with carbon dioxide for cultivation, adjusting the pH again after a period of cultivation, adding ferrous salt to initiate a Fenton reaction, and degrading the organic matter in the wastewater. This method combines biological and chemical methods to treat organic wastewater, utilizing microalgae cultivation combined with Fenton reagent to degrade organic matter, rather than using microalgae alone to treat organic wastewater.

[0007] In summary, for the process of treating organic wastewater using microalgae, appropriate microalgae species should be selected, improved, and cultivated according to the composition of the wastewater to make them suitable for treating the corresponding wastewater, with high treatment efficiency and carbon sequestration at the same time. Summary of the Invention

[0008] To address the problems existing in the prior art, the present invention aims to provide a method for treating organic wastewater and coupling carbon fixation using modified microalgae. The method improves microalgae at the genetic level to increase the expression of cytochrome P450 enzymes, accelerates the primary oxidation of organic pollutants and their entry into the tricarboxylic acid cycle, and promotes the degradation of organic pollutants, thereby achieving the treatment of organic wastewater.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] This invention provides a method for treating organic wastewater and coupling carbon fixation using modified microalgae, the method comprising the following steps:

[0011] (1) Microalgae were cultured under high temperature and high light conditions, and then gene editing technology was used to modify the gene of microalgae cytochrome P450 enzyme to upregulate the expression level of cytochrome P450 enzyme and obtain modified microalgae.

[0012] (2) The improved microalgae obtained in step (1) are subjected to gradient domestication culture in organic wastewater to obtain domesticated microalgae species;

[0013] (3) The microalgae strains that have been domesticated in step (2) are inoculated into the pretreated organic wastewater and cultured in a photobioreactor. During the culture process, the organic matter in the organic wastewater is degraded until the wastewater treatment is completed.

[0014] In this invention, based on the growth characteristics of microalgae, which can fix carbon dioxide during photosynthesis and whose cytochrome P450 enzymes can promote growth and metabolism, the microalgae are genetically modified. The expression of cytochrome P450 enzymes is enhanced through high-temperature and high-light cultivation conditions, accelerating the hydroxylation of aromatic hydrocarbons in organic pollutants, leading to primary oxidation of organic matter and increased polarity, making it more soluble in water and facilitating subsequent metabolic steps. Simultaneously, carbon dioxide is fixed, yielding microalgal biomass. This method can treat various types of organic wastewater with high efficiency and low cost.

[0015] The following are preferred technical solutions of the present invention, but are not intended to limit the technical solutions provided by the present invention. The technical objectives and beneficial effects of the present invention can be better achieved and realized through the following technical solutions.

[0016] As a preferred technical solution of the present invention, the microalgae in step (1) include Scenedesmus or Chlorella.

[0017] Preferably, the microalgae culture conditions in step (1) are as follows: the algal solution temperature is 32–35℃, for example, 32℃, 32.5℃, 33℃, 33.5℃, 34℃, 34.5℃, or 35℃, etc.; the algal solution pH value is 6.5–7.1, for example, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, or 7.1, etc.; and the light intensity is 90–110 μmol·m⁻¹. -2 ·s -1 For example, 90 μmol·m -2 ·s -1 92 μmol·m -2 ·s -1 95 μmol·m -2 ·s -1 98 μmol·m -2 ·s -1 100 μmol·m -2 ·s -1 102 μmol·m -2 ·s -1 105 μmol·m -2 ·s -1108 μmol·m -2 ·s -1 or 110 μmol·m -2 ·s -1 The CO2 concentration in the introduced gas is 5-10%, such as 5%, 6%, 7%, 8%, 9%, or 10%, etc.; but it is not limited to the listed values. Other unlisted values ​​within their respective ranges are also applicable. The CO2 concentration refers to the volume fraction of CO2 in the introduced gas.

[0018] Preferably, during the microalgae culture in step (1), the microalgae are passaged after entering the stable period, and the passage number is more than 3 times, such as 3 times, 4 times, 5 times or 6 times.

[0019] Preferably, the container used for microalgae culture in step (1) includes conical flasks or fermentation flasks, with capacities including 250mL, 300mL, etc.

[0020] As a preferred technical solution of the present invention, the gene editing technology in step (1) is CRISPR-Cas9 gene editing technology.

[0021] Preferably, the gene modification in step (1) is to insert an enhancer into the promoter sequence of the cytochrome P450 enzyme gene to increase the expression level of the cytochrome P450 enzyme.

[0022] Preferably, the expression level of the cytochrome P450 enzyme is upregulated to 3.45 to 5.78 times the original level, for example, 3.45 times, 3.6 times, 3.8 times, 4.0 times, 4.3 times, 4.5 times, 4.8 times, 5.0 times, 5.2 times, 5.5 times, or 5.78 times, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0023] In this invention, the fold increase in the expression level of cytochrome P450 enzymes in microalgae after gene modification is related to the enhancer inserted during gene modification.

[0024] As a preferred technical solution of the present invention, step (2) involves cultivating and acclimatizing improved microalgae in organic wastewater of different concentrations, wherein nutrients required for the growth of microalgae are added to the organic wastewater.

[0025] Preferably, the concentration of the organic wastewater is 50% to 100% of the concentration of the organic wastewater to be treated, such as 50%, 60%, 70%, 80%, 90%, or 100%, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable, and the concentration is selected in a gradient within the range.

[0026] Preferably, the source of the organic wastewater to be treated includes any one or a combination of at least two of the following: cooling tower wastewater from biomass power plants, cooling tower wastewater from coal-fired power plants, or cooling tower wastewater from chemical plants. Typical but non-limiting examples of such combinations include: a combination of cooling tower wastewater from biomass power plants and cooling tower wastewater from coal-fired power plants, a combination of cooling tower wastewater from coal-fired power plants and cooling tower wastewater from chemical plants, and a combination of cooling tower wastewater from biomass power plants, cooling tower wastewater from coal-fired power plants, and cooling tower wastewater from chemical plants, etc.

[0027] Preferably, the COD value of the organic wastewater to be treated is 6 to 10 mg / L, such as 6 mg / L, 6.5 mg / L, 7 mg / L, 7.5 mg / L, 8 mg / L, 8.5 mg / L, 9 mg / L, 9.5 mg / L, or 10 mg / L, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0028] In this invention, taking the cooling tower wastewater of a biomass power plant as an example, the main organic component is benzotriazole, which belongs to nitrogen-containing organic compounds.

[0029] As a preferred technical solution of the present invention, the domestication culture in step (2) is carried out on the plant culture rack in a constant temperature greenhouse, and the culture container used includes conical flasks or fermentation bottles.

[0030] Preferably, the acclimatization and cultivation conditions in step (2) are the same as those in step (1).

[0031] Preferably, step (2) involves domesticating and cultivating microalgae species that can grow in 100% concentration organic wastewater to be treated.

[0032] As a preferred technical solution of the present invention, the pretreatment of organic wastewater in step (3) is to expose the organic wastewater to sunlight for more than 24 hours, such as 24h, 26h, 28h, 30h, 32h, 34h or 36h, but not limited to the listed values. Other unlisted values ​​within this range are also applicable, and air is continuously introduced during the exposure period.

[0033] Preferably, the optical density value (OD680) of the microalgae seed inoculated into the organic wastewater in step (3) is 0.8 to 1, such as 0.8, 0.82, 0.85, 0.88, 0.9, 0.92, 0.95, 0.98 or 1, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0034] In this invention, OD680 refers to the optical density of the microalgae species at a wavelength of 680 nm, also known as the light absorption value, which is a commonly used indicator in microalgae research.

[0035] Preferably, the photobioreactor in step (3) includes any one of a raceway pool, a column photobioreactor, or a horizontal tube photobioreactor.

[0036] As a preferred technical solution of the present invention, the culture conditions of the microalgae in step (3) are as follows: temperature 32-35℃, such as 32℃, 32.5℃, 33℃, 33.5℃, 34℃, 34.5℃ or 35℃, pH value 6-7, such as 6, 6.2, 6.4, 6.5, 6.6, 6.8 or 7, and light intensity 90-110 μmol·m -2 ·s -1 For example, 90 μmol·m -2 ·s -1 92 μmol·m -2 ·s -1 95 μmol·m -2 ·s -1 98 μmol·m -2 ·s -1 100 μmol·m -2 ·s -1 102 μmol·m -2 ·s -1 105 μmol·m -2 ·s -1 108 μmol·m -2 ·s -1 or 110 μmol·m -2 ·s -1 The CO2 concentration in the introduced gas is 5% to 10%, such as 5%, 6%, 7%, 8%, 9%, or 10%, etc.; however, it is not limited to the listed values, and other unlisted values ​​within their respective ranges also apply.

[0037] Preferably, nutrients are added to the photobioreactor during the microalgae cultivation process described in step (3).

[0038] In this invention, the nutrients required for the growth of microalgae mainly include inorganic salts, such as sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, and calcium chloride.

[0039] Preferably, during the microalgae culture process described in step (3), gene expression generates cytochrome P450 enzymes, which accelerate the hydroxylation of organic pollutants and thus promote the degradation of organic pollutants.

[0040] In this invention, the P450 enzyme during the microalgae growth process promotes the degradation of organic matter, such as aromatic hydrocarbons and alkanes. Specifically, for the degradation of aromatic hydrocarbons, the P450 enzyme first promotes oxygen activation, inserting oxygen atoms into the CH bond of the aromatic hydrocarbon to form hydroxyl groups, completing the initial oxidation of the aromatic hydrocarbon. Then, oxygenases open the ring of the aromatic hydrocarbon to form straight-chain compounds, which subsequently enter the tricarboxylic acid cycle and are ultimately oxidized to water and carbon dioxide. For the degradation of straight-chain alkanes, initial oxidation occurs through hydrocarbon oxygenases, followed by further oxidation to fatty acids. These fatty acids are then decomposed into acetyl-CoA via β-oxidation, and acetyl-CoA is completely oxidized to water and carbon dioxide in the tricarboxylic acid cycle.

[0041] Preferably, the culture period of the microalgae in step (3) is 5 to 8 days, such as 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days or 8 days, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0042] As a preferred technical solution of the present invention, the microalgae in step (3) are harvested once for each culture cycle.

[0043] Preferably, the method for harvesting microalgae includes centrifugation and freeze-drying, which consists of two steps: centrifugation and freeze-drying.

[0044] Preferably, the centrifugation speed is 5000 to 7000 rpm, such as 5000 rpm, 5200 rpm, 5500 rpm, 5700 rpm, 6000 rpm, 6200 rpm, 6500 rpm, 6800 rpm or 7000 rpm, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0045] Preferably, the freeze-drying temperature is -50 to -30°C, such as -50°C, -48°C, -45°C, -42°C, -40°C, -38°C, -35°C, -32°C, or -30°C, and the freeze-drying pressure is 0.02 to 0.1 mbar, such as 0.02 mbar, 0.03 mbar, 0.04 mbar, 0.05 mbar, 0.06 mbar, 0.07 mbar, 0.08 mbar, 0.09 mbar, 0.1 mbar, etc., but is not limited to the listed values. Other unlisted values ​​within their respective ranges are also applicable.

[0046] As a preferred technical solution of the present invention, the method includes the following steps:

[0047] (1) The microalgae are cultured under high temperature and high light conditions, wherein the microalgae include Scenedesmus or Chlorella, and the culture conditions are as follows: algal solution temperature is 32-35℃, algal solution pH is 6.5-7.1, and light intensity is 90-110 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas is 5-10%. After the microalgae enter the stable period, they are passaged more than 3 times.

[0048] Then, CRISPR-Cas9 gene editing technology was used to improve the gene of microalgal cytochrome P450 enzyme. The gene improvement method was to insert an enhancer into the promoter sequence of the cytochrome P450 enzyme gene to upregulate the expression level of cytochrome P450 enzyme to 3.45 to 5.78 times the original level, thus obtaining improved microalgae.

[0049] (2) The improved microalgae obtained in step (1) are subjected to gradient acclimatization culture in organic wastewater of different concentrations. The concentration of the organic wastewater is 50-100% of the concentration of the organic wastewater to be treated. The organic wastewater to be treated is selected in a gradient within the concentration range. The source of the organic wastewater to be treated includes any one or at least two of the following: cooling tower wastewater from biomass power plants, cooling tower wastewater from coal-fired power plants, or cooling tower wastewater from chemical plants. The concentration of benzotriazole in the organic wastewater to be treated is 6-10 mg / L. The acclimatization culture conditions are the same as the high temperature and high light culture conditions in step (1). Microalgae species that have been acclimatized and can grow in 100% concentration organic wastewater to be treated are obtained.

[0050] (3) The microalgae strains acclimatized in step (2) are inoculated into pretreated organic wastewater. The pretreatment of the organic wastewater involves exposing it to sunlight for more than 24 hours and continuously purging it with air. The optical density value (OD680) of the microalgae strains after inoculation into the organic wastewater is 0.8–1. The microalgae are then cultured in a photobioreactor, which may be any one of a raceway tank, a column-type photobioreactor, or a horizontal tube-type photobioreactor. The culture conditions for the microalgae are: temperature 32–35℃, pH 6–7, and light intensity 90–110 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas is 5-10%; during the cultivation process, gene expression generates cytochrome P450 enzyme, which accelerates the hydroxylation of organic pollutants and degrades organic pollutants in organic wastewater. The cultivation cycle of the microalgae is 5-8 days until the wastewater treatment is completed; the microalgae are harvested once after each cultivation cycle; the microalgae harvesting method includes centrifugation and freeze-drying, which consists of two steps: centrifugation and freeze-drying. The centrifugation speed is 5000-7000 rrpm, and the freeze-drying temperature is -50 to -30℃, and the pressure is 0.02-0.1 mbar.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The method described in this invention improves microalgae at the gene level and stimulates and regulates them through high temperature and high light cultivation conditions, enhances the expression of cytochrome P450 enzymes, promotes the hydroxylation of organic pollutants, increases polarity, and makes organic pollutants easier to degrade, thereby achieving the effect of treating organic wastewater. At the same time, it can also fix carbon dioxide and obtain microalgae biomass.

[0053] (2) The method described in this invention can treat a variety of types of organic wastewater, with high treatment efficiency and low cost. Detailed Implementation

[0054] To better illustrate the present invention and facilitate understanding of its technical solutions, the present invention is further described in detail below. However, the following embodiments are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0055] The specific embodiments of this invention provide a method for treating organic wastewater and coupling carbon fixation using modified microalgae, the method comprising the following steps:

[0056] (1) Microalgae were cultured under high temperature and high light conditions, and then gene editing technology was used to modify the gene of microalgae cytochrome P450 enzyme to upregulate the expression level of cytochrome P450 enzyme and obtain modified microalgae.

[0057] (2) The improved microalgae obtained in step (1) are subjected to gradient domestication culture in organic wastewater to obtain domesticated microalgae species;

[0058] (3) The microalgae strains that have been domesticated in step (2) are inoculated into the pretreated organic wastewater and cultured in a photobioreactor. During the culture process, the organic matter in the organic wastewater is degraded until the wastewater treatment is completed.

[0059] The following are typical but non-limiting embodiments of the present invention:

[0060] Example 1:

[0061] This embodiment provides a method for treating organic wastewater and coupling carbon fixation using modified microalgae, the method comprising the following steps:

[0062] (1) The microalgae, namely Scenedesmus, were cultured under high temperature and high light conditions. The culture conditions were as follows: algal solution temperature 33℃, algal solution pH 6.8, and light intensity 102 μmol·m⁻¹. -2 ·s -1The CO2 concentration in the introduced gas was 7%, and the microalgae were passaged three times after entering the stationary phase.

[0063] Then, CRISPR-Cas9 gene editing technology was used to improve the gene of microalgal cytochrome P450 enzyme. The gene improvement method was to insert an enhancer into the promoter sequence of the cytochrome P450 enzyme gene to upregulate the expression level of cytochrome P450 enzyme to 4.12 times the original level, thus obtaining improved microalgae.

[0064] (2) The improved microalgae obtained in step (1) are subjected to gradient acclimatization culture in organic wastewater of different concentrations. The concentration gradient of the organic wastewater is selected as 50%, 60%, 70%, 80%, 90%, and 100% of the concentration of the organic wastewater to be treated. The organic wastewater to be treated is the cooling tower wastewater of a biomass power plant, wherein the benzotriazole content is 9.2 mg / L. The acclimatization culture conditions are the same as the high temperature and high light culture conditions in step (1), so as to obtain microalgae species that can grow rapidly in 100% concentration organic wastewater to be treated.

[0065] (3) The microalgae strains acclimated in step (2) are inoculated into the pretreated organic wastewater. The pretreatment of the organic wastewater involves exposing it to sunlight for 30 hours while continuously purging it with air. After inoculation, the microalgae strains have an optical density of 0.85 at a wavelength of 680 nm. They are then cultured in a column-type photobioreactor. The culture conditions for the microalgae are: temperature 33℃, pH 6.5, and light intensity 105 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas is 8%; during the cultivation process, gene expression generates cytochrome P450 enzyme, which accelerates the hydroxylation of organic pollutants and degrades organic pollutants in organic wastewater. The cultivation cycle of the microalgae is 7 days until the wastewater treatment is completed. The microalgae are harvested once per cultivation cycle by centrifugal freeze-drying. The centrifugation speed is 6000 rrpm, the freeze-drying temperature is -40℃, and the pressure is 0.06 mbar.

[0066] In this embodiment, after the above treatment, the content of benzotriazole in the organic wastewater is reduced to 0.94 mg / L, and the removal rate reaches 89.8%; at the same time, 2.77 kg of microalgae powder can be harvested, and based on a carbon content of 50%, the amount of carbon dioxide fixed during the cultivation process is 5.08 kg.

[0067] Example 2:

[0068] This embodiment provides a method for treating organic wastewater and coupling carbon fixation using modified microalgae, the method comprising the following steps:

[0069] (1) The microalgae, namely Scenedesmus, were cultured under high temperature and high light conditions. The culture conditions were as follows: algal solution temperature 32℃, algal solution pH 6.6, and light intensity 95 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas was 6%, and the microalgae were passaged three times after entering the stationary phase.

[0070] Then, CRISPR-Cas9 gene editing technology was used to improve the gene of microalgal cytochrome P450 enzyme. The gene improvement method was to insert an enhancer into the promoter sequence of the cytochrome P450 enzyme gene to upregulate the expression level of cytochrome P450 enzyme to 3.45 times the original level, thus obtaining improved microalgae.

[0071] (2) The improved microalgae obtained in step (1) are subjected to gradient acclimatization culture in organic wastewater of different concentrations. The concentration gradient of the organic wastewater is selected as 50%, 60%, 70%, 80%, 90%, and 100% of the concentration of the organic wastewater to be treated. The organic wastewater to be treated is the cooling tower wastewater of a biomass power plant, wherein the benzotriazole content is 8.83 mg / L. The acclimatization culture conditions are the same as the high temperature and high light culture conditions in step (1), so as to obtain microalgae species that can grow rapidly in 100% concentration organic wastewater to be treated.

[0072] (3) The microalgae strains acclimatized in step (2) are inoculated into the pretreated organic wastewater. The pretreatment of the organic wastewater involves exposing it to sunlight for 25 hours while continuously purging it with air. After inoculation, the microalgae strains have an optical density of 0.81 at a wavelength of 680 nm. They are then cultured in a raceway pond under the following conditions: temperature 32℃, pH 6.2, and light intensity 95 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas is 6%; during the cultivation process, gene expression generates cytochrome P450 enzyme, which accelerates the hydroxylation of organic pollutants and degrades organic pollutants in organic wastewater. The cultivation cycle of the microalgae is 8 days until the wastewater treatment is completed. The microalgae are harvested once per cultivation cycle by centrifugal freeze-drying. The centrifugation speed is 5500 rrpm, the freeze-drying temperature is -45℃, and the pressure is 0.08 mbar.

[0073] In this embodiment, after the above treatment, the content of benzotriazole in the organic wastewater is reduced to 1.23 mg / L, and the removal rate reaches 86.1%; at the same time, 1.24 kg of microalgae powder can be harvested, and based on a carbon content of 50%, the amount of carbon dioxide fixed during the cultivation process is 2.27 kg.

[0074] Example 3:

[0075] This embodiment provides a method for treating organic wastewater and coupling carbon fixation using modified microalgae, the method comprising the following steps:

[0076] (1) The microalgae, namely Scenedesmus, were cultured under high temperature and high light conditions. The culture conditions were as follows: algal solution temperature 35℃, algal solution pH 7.0, and light intensity 109 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas was 9%, and the microalgae were passaged three times after entering the stable period.

[0077] Then, CRISPR-Cas9 gene editing technology was used to improve the gene of microalgal cytochrome P450 enzyme. The gene improvement method was to insert an enhancer into the promoter sequence of the cytochrome P450 enzyme gene to upregulate the expression level of cytochrome P450 enzyme to 5.78 times the original level, thus obtaining improved microalgae.

[0078] (2) The improved microalgae obtained in step (1) are subjected to gradient acclimatization culture in organic wastewater of different concentrations. The concentration gradient of the organic wastewater is selected as 50%, 60%, 70%, 80%, 90%, and 100% of the concentration of the organic wastewater to be treated. The organic wastewater to be treated is the cooling tower wastewater of a biomass power plant, wherein the benzotriazole content is 9.8 mg / L. The acclimatization culture conditions are the same as the high temperature and high light culture conditions in step (1), so as to obtain microalgae species that can grow rapidly in 100% concentration organic wastewater to be treated.

[0079] (3) The microalgae strains acclimated in step (2) are inoculated into the pretreated organic wastewater. The pretreatment of the organic wastewater involves exposing it to sunlight for 28 hours while continuously purging it with air. After inoculation, the microalgae strains have an optical density of 0.95 at a wavelength of 680 nm. They are then cultured in a raceway tank under the following conditions: temperature 34℃, pH 6.5, and light intensity 110 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas is 9%; during the cultivation process, gene expression generates cytochrome P450 enzyme, which accelerates the hydroxylation of organic pollutants and degrades organic pollutants in organic wastewater. The cultivation cycle of the microalgae is 6 days until the wastewater treatment is completed. The microalgae are harvested once per cultivation cycle by centrifugal freeze-drying. The centrifugation speed is 6500 rrpm, the freeze-drying temperature is -35℃, and the pressure is 0.04 mbar.

[0080] In this embodiment, after the above treatment, the content of benzotriazole in the organic wastewater is reduced to 0.92 mg / L, and the removal rate reaches 90.6%; at the same time, 4.54 kg of microalgae powder can be harvested, and based on a carbon content of 50%, the amount of carbon dioxide fixed during the cultivation process is 8.32 kg.

[0081] Example 4:

[0082] This embodiment provides a method for treating organic wastewater and coupling carbon fixation using modified microalgae, the method comprising the following steps:

[0083] (1) The microalgae, namely Chlorella vulgaris, were cultured under high temperature and high light conditions. The culture conditions were as follows: algal solution temperature 34℃, algal solution pH 6.6, and light intensity 105 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas was 10%, and the microalgae were passaged four times after entering the stationary phase.

[0084] Then, CRISPR-Cas9 gene editing technology was used to improve the gene of microalgal cytochrome P450 enzyme. The gene improvement method was to insert an enhancer into the promoter sequence of the cytochrome P450 enzyme gene to upregulate the expression level of cytochrome P450 enzyme to 5.05 times the original level, thus obtaining improved microalgae.

[0085] (2) The improved microalgae obtained in step (1) are subjected to gradient acclimatization culture in organic wastewater of different concentrations. The concentration gradient of the organic wastewater is selected as 50%, 58%, 66%, 74%, 82%, 90%, and 100% of the concentration of the organic wastewater to be treated. The organic wastewater to be treated is the cooling tower wastewater of a biomass power plant, wherein the benzotriazole content is 7.6 mg / L. The acclimatization culture conditions are the same as the high temperature and high light culture conditions in step (1), so as to obtain microalgae species that can grow rapidly in 100% concentration organic wastewater to be treated.

[0086] (3) The microalgae strains acclimated in step (2) are inoculated into the pretreated organic wastewater. The pretreatment of the organic wastewater involves exposing it to sunlight for 26 hours while continuously purging it with air. After inoculation, the microalgae strains have an optical density of 0.9 at a wavelength of 680 nm and are cultured in a horizontal tube photobioreactor. The culture conditions for the microalgae are: temperature 35℃, pH 7.0, and light intensity 101 μmol·m⁻¹. -2 ·s -1The CO2 concentration in the introduced gas is 8.5%; during the cultivation process, gene expression generates cytochrome P450 enzyme, which accelerates the hydroxylation of organic pollutants and degrades organic pollutants in organic wastewater. The cultivation cycle of the microalgae is 6 days until the wastewater treatment is completed. The microalgae are harvested once per cultivation cycle by centrifugal freeze-drying. The centrifugation speed is 7000 rrpm, the freeze-drying temperature is -30℃, and the pressure is 0.02 mbar.

[0087] In this embodiment, after the above treatment, the content of benzotriazole in the organic wastewater is reduced to 0.92 mg / L, and the removal rate reaches 87.9%; at the same time, 3.27 kg of microalgae powder can be harvested, and based on a carbon content of 50%, the amount of carbon dioxide fixed during the cultivation process is 5.99 kg.

[0088] Example 5:

[0089] This embodiment provides a method for treating organic wastewater and coupling carbon fixation using modified microalgae, the method comprising the following steps:

[0090] (1) The microalgae, namely Scenedesmus, were cultured under high temperature and high light conditions. The culture conditions were as follows: algal solution temperature 33.5℃, algal solution pH 7.1, and light intensity 92 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas is 5%, and the microalgae are passaged 4 times after entering the stationary phase.

[0091] Then, the gene of microalgal cytochrome P450 enzyme was modified using CRISPR-Cas9 gene editing technology. The gene modification method was to insert an enhancer into the promoter sequence of the cytochrome P450 enzyme gene, thereby upregulating the expression level of cytochrome P450 enzyme to 4.50 times the original level, thus obtaining modified microalgae.

[0092] (2) The improved microalgae obtained in step (1) are subjected to gradient acclimatization culture in organic wastewater of different concentrations. The concentration gradient of the organic wastewater is selected as 50%, 62%, 74%, 86%, 98%, and 100% of the concentration of the organic wastewater to be treated. The organic wastewater to be treated is the cooling tower wastewater of a coal-fired power plant, wherein the benzotriazole content is 6.9 mg / L. The acclimatization culture conditions are the same as the high temperature and high light culture conditions in step (1), so as to obtain microalgae species that can grow rapidly in 100% concentration organic wastewater to be treated.

[0093] (3) The microalgae strains acclimated in step (2) are inoculated into the pretreated organic wastewater. The pretreatment of the organic wastewater involves exposing it to sunlight for 24 hours while continuously purging it with air. After inoculation, the microalgae strains have an optical density of 1.0 at a wavelength of 680 nm and are cultured in a horizontal tube photobioreactor. The culture conditions for the microalgae are: temperature 32.5℃, pH 6.6, and light intensity 93 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas is 5.5%; during the cultivation process, gene expression generates cytochrome P450 enzyme, which accelerates the hydroxylation of organic pollutants and degrades organic pollutants in organic wastewater. The cultivation cycle of the microalgae is 6.5 days until the wastewater treatment is completed. The microalgae are harvested once after each cultivation cycle. The microalgae are harvested by centrifugal freeze-drying. The centrifugation speed is 5000 rrpm, the freeze-drying temperature is -50℃, and the pressure is 0.1 mbar.

[0094] In this embodiment, after the above treatment, the content of benzotriazole in the organic wastewater is reduced to 0.57 mg / L, and the removal rate reaches 91.7%; at the same time, 2.3 kg of microalgae powder can be harvested, and based on a carbon content of 50%, the amount of carbon dioxide fixed during the cultivation process is 4.22 kg.

[0095] In summary, the above embodiments demonstrate that the method of the present invention improves microalgae at the genetic level and enhances the expression of cytochrome P450 enzymes through stimulation and regulation under high temperature and high light cultivation conditions. This promotes the hydroxylation of organic pollutants, increases their polarity, and makes them easier to degrade, thereby treating organic wastewater. Simultaneously, it can fix carbon dioxide and obtain microalgal biomass. The method can treat various types of organic wastewater with high efficiency and low cost.

[0096] The applicant declares that the present invention is illustrated through the above embodiments, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for the methods of the present invention, additions of auxiliary steps, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for treating organic wastewater and coupling carbon fixation using modified microalgae, characterized in that, The method includes the following steps: (1) Microalgae were cultured under high temperature and high light conditions, and then the gene editing technology was used to modify the gene of microalgae cytochrome P450 enzyme. The expression level of cytochrome P450 enzyme was upregulated to 3.45~5.78 times the original level, and the modified microalgae were obtained. The microalgae culture conditions described in step (1) are as follows: algal solution temperature 32~35℃, algal solution pH 6.5~7.1, and light intensity 90~110 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas is 5-10%; The method of modifying the gene in step (1) is to insert an enhancer into the promoter sequence of the cytochrome P450 enzyme gene to increase the expression level of the cytochrome P450 enzyme. (2) The improved microalgae obtained in step (1) are subjected to gradient domestication culture in organic wastewater to obtain domesticated microalgae species; The concentration of the organic wastewater is 50-100% of the concentration of the organic wastewater to be treated, and is selected in a gradient within the concentration range. The concentration of benzotriazole in the organic wastewater to be treated is 6~10 mg / L; (3) The microalgae strains that have been domesticated in step (2) are inoculated into the pretreated organic wastewater and cultured in a photobioreactor. During the culture process, the organic matter in the organic wastewater is degraded until the wastewater treatment is completed.

2. The method according to claim 1, characterized in that, The microalgae mentioned in step (1) include Scenedesmus or Chlorella.

3. The method according to claim 1, characterized in that, When culturing microalgae in step (1), the microalgae are passaged after entering the stable period, and the passage number is more than 3 times.

4. The method according to claim 1, characterized in that, The containers used for microalgae cultivation in step (1) include conical flasks or fermentation flasks.

5. The method according to claim 1, characterized in that, The gene editing technology described in step (1) is CRISPR-Cas9 gene editing technology.

6. The method according to claim 1, characterized in that, Step (2) Cultivate and acclimatize improved microalgae in organic wastewater of different concentrations, and add nutrients required for microalgae growth to the organic wastewater.

7. The method according to claim 1, characterized in that, The sources of the organic wastewater to be treated include any one or a combination of at least two of the following: cooling tower wastewater from biomass power plants, cooling tower wastewater from coal-fired power plants, or cooling tower wastewater from chemical plants.

8. The method according to claim 1, characterized in that, The domestication culture described in step (2) is carried out on plant culture racks in a constant temperature greenhouse, and the culture containers used include conical flasks or fermentation bottles.

9. The method according to claim 1, characterized in that, The conditions for domestication and cultivation in step (2) are the same as those in step (1).

10. The method according to claim 1, characterized in that, Step (2) Domestication and cultivation of microalgae species that can grow in 100% concentration of organic wastewater to be treated.

11. The method according to claim 1, characterized in that, The pretreatment of the organic wastewater in step (3) involves exposing the organic wastewater to sunlight for more than 24 hours and continuously introducing air.

12. The method according to claim 1, characterized in that, The optical density value after the microalgae strains in step (3) are inoculated into the organic wastewater is 0.8~1.

13. The method according to claim 1, characterized in that, The photobioreactor in step (3) includes any one of a raceway pool, a column photobioreactor, or a horizontal tube photobioreactor.

14. The method according to claim 1, characterized in that, The culture conditions for the microalgae in step (3) are: temperature 32~35℃, pH 6~7, and light intensity 90~110 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas is 5-10%.

15. The method according to claim 1, characterized in that, In step (3), nutrients are added to the photobioreactor during the microalgae cultivation process.

16. The method according to claim 1, characterized in that, In step (3), during the microalgae culture process, gene expression generates cytochrome P450 enzymes, which accelerate the hydroxylation of organic pollutants and thus promote the degradation of organic pollutants.

17. The method according to claim 1, characterized in that, The culture period for the microalgae in step (3) is 5 to 8 days.

18. The method according to claim 1, characterized in that, In step (3), the microalgae are harvested once for each culture cycle.

19. The method according to claim 18, characterized in that, The method for harvesting microalgae includes centrifugation and freeze-drying, which consists of two steps: centrifugation and freeze-drying.

20. The method according to claim 19, characterized in that, The centrifuge speed is 5000~7000 rpm.

21. The method according to claim 19, characterized in that, The freeze-drying temperature is -50 to -30°C, and the freeze-drying pressure is 0.02 to 0.1 mbar.

22. The method according to claim 1, characterized in that, The method includes the following steps: (1) The microalgae are cultured under high temperature and high light conditions, wherein the microalgae include Scenedesmus or Chlorella, and the culture conditions are as follows: algal solution temperature is 32~35℃, algal solution pH is 6.5~7.1, and light intensity is 90~110 μmol·m -2 ·s -1 The CO2 concentration in the introduced gas is 5-10%. After the microalgae enter the stable period, they are passaged more than 3 times. Then, the gene of microalgal cytochrome P450 enzyme was modified using CRISPR-Cas9 gene editing technology. The gene was modified by inserting an enhancer into the promoter sequence of the cytochrome P450 enzyme gene, which upregulated the expression level of cytochrome P450 enzyme to 3.45 to 5.78 times the original level, thus obtaining modified microalgae. (2) The improved microalgae obtained in step (1) are subjected to gradient acclimatization culture in organic wastewater of different concentrations. The concentration of the organic wastewater is 50-100% of the concentration of the organic wastewater to be treated. The organic wastewater to be treated is selected in a gradient within the concentration range. The source of the organic wastewater to be treated includes any one or at least two of the following: cooling tower wastewater from biomass power plants, cooling tower wastewater from coal-fired power plants, or cooling tower wastewater from chemical plants. The concentration of benzotriazole in the organic wastewater to be treated is 6-10 mg / L. The acclimatization culture conditions are the same as the high temperature and high light culture conditions in step (1). Microalgae species that have been acclimatized and can grow in 100% concentration organic wastewater to be treated are obtained. (3) The microalgae strains that have been domesticated in step (2) are inoculated into the pretreated organic wastewater. The pretreatment of the organic wastewater involves exposing it to sunlight for more than 24 hours and continuously purging it with air. The light density of the microalgae strains after inoculation into the organic wastewater is 0.8 to 1. The microalgae are then cultured in a photobioreactor, which includes any one of a raceway pool, a column photobioreactor, or a horizontal tube photobioreactor. The culture conditions for the microalgae are: temperature 32 to 35°C, pH 6 to 7, and light intensity 90 to 110 μmol·m⁻¹. -2 ·s -1 The CO2 concentration in the introduced gas is 5-10%; during the cultivation process, gene expression generates cytochrome P450 enzyme, which accelerates the hydroxylation of organic pollutants and degrades organic pollutants in organic wastewater. The cultivation cycle of the microalgae is 5-8 days until the wastewater treatment is completed; the microalgae are harvested once after each cultivation cycle; the microalgae harvesting method includes centrifugation and freeze-drying, which consists of two steps: centrifugation and freeze-drying. The centrifugation speed is 5000-7000 rpm, and the freeze-drying temperature is -50 to -30℃, and the pressure is 0.02-0.1 mbar.