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

Abstract

The invention provides a method for treating organic wastewater and coupling carbon sequestration by utilizing improved microalgae, which comprises the following steps: culturing the microalgae under high-temperature and high-light conditions, improving the gene of microalgae cytochrome P450 enzyme by adopting a gene editing technology, and increasing the expression quantity of the cytochrome P450 enzyme; the method comprises the following steps: carrying out gradient domestication culture on improved microalgae in organic wastewater, inoculating the domesticated microalgae into the pretreated organic wastewater, culturing in a photobioreactor, and degrading organic matters in the wastewater in the culture process. Microalgae are improved from the gene level, the expression quantity of cytochrome P450 enzyme is strengthened and hydroxylation of organic pollutants is promoted through stimulation, regulation and control of high-temperature and high-light culture conditions, so that the organic pollutants are easier to degrade, and carbon dioxide can be fixed; the method can be used for treating various types of organic wastewater, and is high in treatment efficiency and relatively low in cost.

Description

Technical Field

[0001] The invention belongs to the technical field of organic wastewater treatment, and relates to a method for treating organic wastewater by utilizing improved microalgae and coupling carbon fixation. Background Art

[0002] With the development of society and human production activities, pollution problems are inevitable. In order to avoid wastewater pollution, wastewater treatment is required before discharge. Organic wastewater has become one of the difficulties in wastewater treatment due to its difficult-to-degrade characteristics. Its sources include chemical, pharmaceutical, fuel, food, electricity and many other production fields. In addition to carbon, organic wastewater often contains nitrogen and phosphorus, which are important factors causing eutrophication of water bodies. Therefore, the treatment of organic pollutants in wastewater usually requires degrading them to generate small molecular harmless substances and realizing the transformation and utilization of pollutants in wastewater as much as possible.

[0003] Take biomass power plants as an example. The combustion of biomass is used to generate steam for power generation. The steam is cooled by water in the cooling tower. In order to reduce the scaling and corrosion of the coils in the cooling tower, it is necessary to add scale inhibitors to the circulating water of the cooling tower. Usually, they are organic compounds, and benzotriazole is one of the effective ingredients. In order to maintain the quality of the circulating water in the cooling tower, it is necessary to discharge and replenish the sewage regularly. The sewage usually contains inorganic salts, humus and organic pollutants. Organic pollutants are easy to cause toxic effects on aquatic organisms, and scaling will occur during the desalination process, affecting the performance and life of the system. Therefore, it is necessary to treat the sewage from the cooling tower, especially the treatment of organic pollutants.

[0004] The treatment of organic pollutants can be roughly divided into physical, chemical and biological methods. Physical means include microfiltration, ultrafiltration, coagulation, adsorption, etc., which often have disadvantages such as low removal efficiency and long time consumption. Traditional chemical methods such as precipitation and oxidation are difficult to treat organic matter, so electrochemical oxidation can be used, but its cost is relatively high and it is difficult to treat large amounts of wastewater. Biological methods have the advantages of eco-friendliness, resource recovery, low energy consumption, and low cost, and have broad application prospects.

[0005] Microalgae refers to algae groups whose morphology can only be distinguished under a microscope. They have a good treatment effect on organic wastewater, can fix carbon dioxide through photosynthesis, and accumulate nutrients such as protein and oil. CN 105621783A discloses a method for removing nitrogen and phosphorus from sewage using a sequencing batch microalgae reactor, the method comprising: pretreatment of nitrogen- and phosphorus-rich sewage; inoculating microalgae in a sequencing batch microalgae reactor, introducing pretreated sewage into the microalgae reactor, controlling the volume exchange rate according to the total nitrogen content in the sewage, turning on an external light source, aeration reaction, and after the reaction, the algae liquid is allowed to settle, the supernatant is discharged, and sewage is repeatedly introduced for treatment as needed, and algae mud is discharged regularly. In this method, microalgae are used instead of activated sludge as the reaction subject to achieve the removal of nitrogen and phosphorus in wastewater, but the nitrogen and phosphorus in the wastewater exist in the form of inorganic salts, which are consumed as nutrients for photosynthesis rather than degrading nitrogen-containing organic matter.

[0006] CN 109368801A discloses a method for treating organic pollutants in water by a photo-assisted Fenton system based on microalgae, which comprises: inoculating microalgae in wastewater, adjusting pH, setting temperature, light intensity and light time, introducing air mixed with carbon dioxide for cultivation, adjusting pH again after cultivation for a period of time, adding ferrous salt for Fenton reaction, and degrading organic matter in wastewater. The method is to treat organic wastewater by combining biological method and chemical method, and degrade organic matter by using microalgae cultivation combined with Fenton reagent, rather than using microalgae alone to treat organic wastewater.

[0007] In summary, for the process of using microalgae to treat organic wastewater, according to the composition of the wastewater, suitable microalgae species are selected for improvement and cultivation, so that they are suitable for treating the corresponding wastewater, have high treatment efficiency, and at the same time play a role in carbon fixation. Summary of the invention

[0008] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for treating organic wastewater using modified microalgae and coupling carbon fixation. The method improves the microalgae at the genetic level, increases the expression of cytochrome P450 enzymes, accelerates the primary oxidation of organic pollutants, enters the tricarboxylic acid cycle and other metabolic processes, promotes the degradation of organic pollutants, and achieves the treatment of organic wastewater.

[0009] To achieve this object, the present invention adopts the following technical solutions:

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

[0011] (1) Cultivating microalgae under high temperature and high light conditions, and then using gene editing technology to improve the gene of cytochrome P450 enzyme of the microalgae, thereby increasing the expression level of cytochrome P450 enzyme and obtaining improved microalgae;

[0012] (2) culturing the modified microalgae obtained in step (1) in a gradient manner in organic wastewater to obtain domesticated microalgae species;

[0013] (3) inoculating the microalgae species domesticated in step (2) into the pretreated organic wastewater and culturing them in a photobioreactor, wherein the organic matter in the organic wastewater is degraded during the culturing process until the wastewater treatment is completed.

[0014] In the present invention, based on the growth characteristics of microalgae, it can fix carbon dioxide during photosynthesis, and the cytochrome P450 enzyme contained in it can play a good promoting role in the growth and metabolism of microalgae. Therefore, in the present invention, the microalgae is improved at the genetic level, and the expression of cytochrome P450 enzyme is enhanced through high temperature and high light culture conditions stimulation and regulation, the hydroxylation reaction of aromatic hydrocarbons in organic pollutants is accelerated, the organic matter is primary oxidized and the polarity is increased, so that it is easily soluble in water, which is convenient for subsequent metabolic steps; at the same time, carbon dioxide can also be fixed to obtain microalgae biomass; the method can treat various types of organic wastewater, with high treatment efficiency and low cost.

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

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

[0017] Preferably, the conditions for culturing the microalgae in step (1) are as follows: the algae solution temperature is 32-35°C, such as 32°C, 32.5°C, 33°C, 33.5°C, 34°C, 34.5°C or 35°C, the algae solution pH is 6.5-7.1, such as 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 or 7.1, and the light intensity is 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 etc., CO in the introduced gas 2 The concentration is 5-10%, for example 5%, 6%, 7%, 8%, 9% or 10%, etc.; but it is not limited to the listed values, and other unlisted values ​​within the respective numerical ranges are also applicable. 2 The concentration refers to the CO in the gas. 2 volume fraction.

[0018] Preferably, during the culturing of the microalgae in step (1), the microalgae are subcultured after entering a stable phase, and the number of subcultures is 3 or more times, for example, 3, 4, 5 or 6 times.

[0019] Preferably, the container used for the microalgae culture in step (1) includes a conical flask or a fermentation flask, and its specification capacity includes 250 mL, 300 mL, 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 performed by inserting an enhancer into the gene promoter sequence of the cytochrome P450 enzyme 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 of 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, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

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

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

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

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

[0027] Preferably, the COD value of the organic wastewater to be treated is 6 to 10 mg / L, for example, 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, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0028] In the present invention, when taking the cooling tower wastewater of a biomass power plant as an example, the organic component thereof is mainly benzotriazole, which is a nitrogen-containing organic matter.

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

[0030] Preferably, the conditions for the acclimation culture in step (2) are the same as the culture conditions in step (1).

[0031] Preferably, in step (2), microalgae species are domesticated and cultured that can grow in 100% concentration of the organic wastewater to be treated.

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

[0033] Preferably, the optical density value (OD680) of the microalgae species after inoculation into the organic wastewater in step (3) is 0.8 to 1, for example, 0.8, 0.82, 0.85, 0.88, 0.9, 0.92, 0.95, 0.98 or 1, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

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

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

[0036] As a preferred technical solution of the present invention, the culture conditions of the microalgae in step (3) are: a temperature of 32 to 35°C, such as 32°C, 32.5°C, 33°C, 33.5°C, 34°C, 34.5°C or 35°C, a pH of 6 to 7, such as 6, 6.2, 6.4, 6.5, 6.6, 6.8 or 7, and a light intensity of 90 to 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 etc., CO in the introduced gas 2 The concentration is 5-10%, for example 5%, 6%, 7%, 8%, 9% or 10%, etc.; but it is not limited to the listed values, and other unlisted values ​​within the respective numerical ranges are also applicable.

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

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

[0039] Preferably, during the microalgae cultivation process 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 the present invention, the P450 enzyme in the growth process of the microalgae promotes the degradation of organic matter, such as the degradation of aromatic hydrocarbons and alkanes; wherein, for the degradation of aromatic hydrocarbons, the P450 enzyme first promotes the activation of oxygen, inserts the oxygen atom into the CH bond of the aromatic hydrocarbon to form a hydroxyl group, completes the initial oxidation of the aromatic hydrocarbon, and then opens the aromatic hydrocarbon ring through the oxygenase to form a straight-chain compound, which then enters the tricarboxylic acid cycle and is finally oxidized to water and carbon dioxide; for the degradation of straight-chain alkanes, the hydrocarbon oxygenase is used for initial oxidation, and then further oxidized to fatty acids, which are then decomposed into acetyl-CoA through the β-oxidation pathway, 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, for example, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days or 8 days, but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

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

[0043] Preferably, the method for harvesting microalgae includes a centrifugal freeze-drying method, which is divided into two steps: centrifugation and freeze-drying.

[0044] Preferably, the centrifugal rotation speed is 5000-7000rpm, for example 5000rpm, 5200rpm, 5500rpm, 5700rpm, 6000rpm, 6200rpm, 6500rpm, 6800rpm or 7000rpm, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0045] Preferably, the freeze-drying temperature is -50 to -30°C, for example, -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, for example, 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 are not limited to the listed values, and other unlisted values ​​within the respective numerical ranges are equally applicable.

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

[0047] (1) Cultivating microalgae under high temperature and high light conditions, wherein the microalgae include Scenedesmus or Chlorella, and the conditions for culturing the microalgae are: an algae liquid temperature of 32 to 35° C., an algae liquid pH value of 6.5 to 7.1, and a light intensity of 90 to 110 μmol·m -2 ·s -1 , CO in the gas introduced 2 The concentration is 5-10%, and the microalgae are subcultured after entering the stable period, and the number of subcultures is more than 3 times;

[0048] Then, the CRISPR-Cas9 gene editing technology is used to improve the gene of the microalgae cytochrome P450 enzyme, wherein the gene improvement method is to insert an enhancer into the gene promoter sequence of the cytochrome P450 enzyme to increase the expression level of the cytochrome P450 enzyme to 3.45 to 5.78 times of the original, thereby obtaining improved microalgae;

[0049] (2) The modified microalgae obtained in step (1) are subjected to gradient acclimation and culture in organic wastewater of different concentrations, wherein the concentration of the organic wastewater is 50-100% of the concentration of the organic wastewater to be treated, and the gradient is selected within the concentration range. The source of the organic wastewater to be treated includes any one or a combination of at least two of the cooling tower wastewater of a biomass power plant, the cooling tower wastewater of a coal-fired power plant, or the cooling tower wastewater of a chemical plant. The concentration of benzotriazole in the organic wastewater to be treated is 6-10 mg / L. The conditions of the acclimation and culture are the same as the high temperature and high light culture conditions in step (1), so as to obtain acclimated microalgae species capable of growing in 100% concentration of the organic wastewater to be treated;

[0050] (3) inoculating the microalgae species acclimated in step (2) into pretreated organic wastewater, wherein the pretreatment of the organic wastewater is to expose the organic wastewater to light for more than 24 hours and continuously introduce air; the optical density (OD680) of the microalgae species after inoculation into the organic wastewater is 0.8 to 1, and the microalgae species are cultured in a photobioreactor, wherein the photobioreactor includes any one of a raceway pool, a column-type photobioreactor, or a horizontal tube-type photobioreactor, and the culture conditions of the microalgae are: a temperature of 32 to 35° C., a pH of 6 to 7, and a light intensity of 90 to 110 μmol·m -2 ·s -1 , CO in the gas introduced 2The concentration is 5 to 10%; during the culture process, gene expression generates cytochrome P450 enzyme, which accelerates the hydroxylation of organic pollutants and degrades organic pollutants in organic wastewater. The culture cycle of the microalgae is 5 to 8 days until the wastewater treatment is completed; the microalgae are harvested once after each culture cycle; the method for harvesting the microalgae includes centrifugal freeze-drying, which is divided into two steps of centrifugation and freeze-drying, the centrifugal speed is 5000 to 7000 rrpm, the freeze-drying temperature is -50 to -30°C, and the pressure is 0.02 to 0.1 mbar.

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

[0052] (1) The method of the present invention improves the microalgae at the genetic level, and through high temperature and high light culture conditions, stimulates and regulates, strengthens the expression of cytochrome P450 enzymes, promotes the hydroxylation of organic pollutants, increases polarity, and makes organic pollutants more easily degraded, thereby achieving the effect of treating organic wastewater, and can also fix carbon dioxide and obtain microalgae biomass;

[0053] (2) The method of the present invention can treat various types of organic wastewater with high treatment efficiency and low cost. DETAILED DESCRIPTION

[0054] In order to better illustrate the present invention and facilitate understanding of the technical solution of the present invention, the present invention is further described in detail below. However, the following embodiments are only simple 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 shall be subject to the claims.

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

[0056] (1) Cultivating microalgae under high temperature and high light conditions, and then using gene editing technology to improve the gene of cytochrome P450 enzyme of the microalgae, thereby increasing the expression level of cytochrome P450 enzyme and obtaining improved microalgae;

[0057] (2) culturing the modified microalgae obtained in step (1) in a gradient manner in organic wastewater to obtain domesticated microalgae species;

[0058] (3) inoculating the microalgae species domesticated in step (2) into the pretreated organic wastewater and culturing them in a photobioreactor, wherein the organic matter in the organic wastewater is degraded during the culturing process until the wastewater treatment is completed.

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

[0060] Embodiment 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) Cultivating microalgae under high temperature and high light conditions, wherein the microalgae is Scenedesmus, and the conditions for culturing the microalgae are: an algae liquid temperature of 33° C., an algae liquid pH value of 6.8, and a light intensity of 102 μmol·m -2 ·s -1 , CO in the gas introduced 2 The concentration was 7%, and the microalgae were subcultured after entering the stable phase, and the subculture was repeated three times;

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

[0064] (2) The modified microalgae obtained in step (1) are subjected to gradient acclimation and culture in organic wastewater of different concentrations, wherein the concentration gradient of the organic wastewater is selected to be 50%, 60%, 70%, 80%, 90%, and 100% of the concentration of the organic wastewater to be treated, respectively; the organic wastewater to be treated is cooling tower wastewater from a biomass power plant, wherein the content of benzotriazole is 9.2 mg / L; the conditions of the acclimation and culture are the same as the high temperature and high light culture conditions in step (1), and a microalgae species capable of growing rapidly in 100% concentration of organic wastewater to be treated is obtained;

[0065] (3) inoculating the microalgae species acclimated in step (2) into the pretreated organic wastewater, wherein the pretreatment of the organic wastewater is to expose the organic wastewater to light for 30 hours, during which air is continuously introduced; after the microalgae species are inoculated into the organic wastewater, the optical density at a wavelength of 680 nm is 0.85, and the microalgae are cultured in a column-type photobioreactor, and the culture conditions of the microalgae are: temperature of 33° C., pH of 6.5, and light intensity of 105 μmol·m -2 ·s -1 , CO in the gas introduced 2 The concentration is 8%; during the culture process, the gene expression generates cytochrome P450 enzyme, which accelerates the hydroxylation of organic pollutants and degrades organic pollutants in organic wastewater. The culture cycle of the microalgae is 7 days until the wastewater treatment is completed; the microalgae is harvested once after each culture cycle, and the method for harvesting the microalgae is centrifugal freeze-drying, the centrifugal speed is 6000rrpm, the freeze-drying temperature is -40°C, and the pressure is 0.06mbar.

[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. Calculated based on the carbon content of 50%, the amount of carbon dioxide fixed during the cultivation process is 5.08 kg.

[0067] Embodiment 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) Cultivating microalgae under high temperature and high light conditions, wherein the microalgae is Scenedesmus, and the conditions for culturing the microalgae are: an algae solution temperature of 32° C., an algae solution pH value of 6.6, and a light intensity of 95 μmol·m -2 ·s -1 , CO in the gas introduced 2 The concentration was 6%, and the microalgae were subcultured after entering the stable phase, and the subculture was repeated three times;

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

[0071] (2) The modified microalgae obtained in step (1) are subjected to gradient acclimation and culture in organic wastewater of different concentrations, wherein the concentration gradient of the organic wastewater is selected to be 50%, 60%, 70%, 80%, 90%, and 100% of the concentration of the organic wastewater to be treated, respectively; the organic wastewater to be treated is cooling tower wastewater from a biomass power plant, wherein the content of benzotriazole is 8.83 mg / L; the conditions of the acclimation and culture are the same as the high temperature and high light culture conditions in step (1), and a microalgae species capable of growing rapidly in 100% concentration of organic wastewater to be treated is obtained;

[0072] (3) inoculating the microalgae species acclimated in step (2) into the pretreated organic wastewater, wherein the pretreatment of the organic wastewater is to expose the organic wastewater to light for 25 hours, during which air is continuously introduced; after the microalgae species are inoculated into the organic wastewater, the optical density at a wavelength of 680 nm is 0.81, and the microalgae are cultured in a runway pool, and the culture conditions of the microalgae are: temperature of 32° C., pH of 6.2, and light intensity of 95 μmol·m -2 ·s -1 , CO in the gas introduced 2The concentration is 6%; during the culture process, the gene expression generates cytochrome P450 enzyme, which accelerates the hydroxylation of organic pollutants and degrades organic pollutants in organic wastewater. The culture cycle of the microalgae is 8 days until the wastewater treatment is completed; the microalgae is harvested once after each culture cycle, and the method for harvesting the microalgae is centrifugal freeze-drying, the centrifugal speed is 5500rrpm, the freeze-drying temperature is -45°C, and the pressure is 0.08mbar.

[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. Calculated based on the carbon content of 50%, the amount of carbon dioxide fixed during the cultivation process is 2.27 kg.

[0074] Embodiment 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) Cultivating microalgae under high temperature and high light conditions, wherein the microalgae is Scenedesmus, and the conditions for culturing the microalgae are: an algae solution temperature of 35° C., an algae solution pH value of 7.0, and a light intensity of 109 μmol·m -2 ·s -1 , CO in the gas introduced 2 The concentration was 9%, and the microalgae were subcultured after entering the stable phase, and the subculture was repeated three times;

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

[0078] (2) The modified microalgae obtained in step (1) are subjected to gradient acclimation and culture in organic wastewater of different concentrations, wherein the concentration gradient of the organic wastewater is selected to be 50%, 60%, 70%, 80%, 90%, and 100% of the concentration of the organic wastewater to be treated, respectively; the organic wastewater to be treated is cooling tower wastewater from a biomass power plant, wherein the benzotriazole content is 9.8 mg / L; the conditions of the acclimation and culture are the same as the high temperature and high light culture conditions in step (1), and a microalgae species capable of growing rapidly in 100% concentration of organic wastewater to be treated is obtained;

[0079] (3) inoculating the microalgae species acclimated in step (2) into the pretreated organic wastewater, wherein the pretreatment of the organic wastewater is to expose the organic wastewater to light for 28 hours, during which air is continuously introduced; after the microalgae species are inoculated into the organic wastewater, the optical density at a wavelength of 680 nm is 0.95, and the microalgae are cultured in a runway pool, and the culture conditions of the microalgae are: temperature of 34° C., pH of 6.5, and light intensity of 110 μmol·m -2 ·s -1 , CO in the gas introduced 2 The concentration is 9%; during the cultivation process, the 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 is harvested once after each cultivation cycle, and the method for harvesting the microalgae is centrifugal freeze-drying, the centrifugal speed is 6500rrpm, the freeze-drying temperature is -35°C, and the pressure is 0.04mbar.

[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. According to the carbon content of 50%, the amount of carbon dioxide fixed during the cultivation process is 8.32 kg.

[0081] Embodiment 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) Cultivating microalgae under high temperature and high light conditions, wherein the microalgae is Chlorella vulgaris, and the conditions for culturing the microalgae are: an algae liquid temperature of 34° C., an algae liquid pH value of 6.6, and a light intensity of 105 μmol·m -2 ·s -1 , CO in the gas introduced 2 The concentration was 10%, and the microalgae were subcultured after entering the stable phase, and the subculture was repeated 4 times;

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

[0085] (2) The modified microalgae obtained in step (1) are subjected to gradient acclimation and culture in organic wastewater of different concentrations, wherein the concentration gradient of the organic wastewater is selected to be 50%, 58%, 66%, 74%, 82%, 90%, and 100% of the concentration of the organic wastewater to be treated, respectively; the organic wastewater to be treated is cooling tower wastewater from a biomass power plant, wherein the benzotriazole content is 7.6 mg / L; the conditions of the acclimation and culture are the same as the high temperature and high light culture conditions in step (1), and a microalgae species capable of growing rapidly in 100% concentration of organic wastewater to be treated is obtained;

[0086] (3) inoculating the microalgae species acclimated in step (2) into the pretreated organic wastewater, wherein the pretreatment of the organic wastewater is to expose the organic wastewater to light for 26 hours, during which air is continuously introduced; after the microalgae species are inoculated into the organic wastewater, the optical density at a wavelength of 680 nm is 0.9, and the microalgae are cultured in a horizontal tube photobioreactor, and the culture conditions of the microalgae are: temperature of 35° C., pH of 7.0, and light intensity of 101 μmol·m -2 ·s -1 , CO in the gas introduced 2 The concentration is 8.5%; during the cultivation process, the 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 is harvested once after each cultivation cycle, and the method for harvesting the microalgae is centrifugal freeze-drying, the centrifugal speed is 7000rrpm, the freeze-drying temperature is -30°C, and the pressure is 0.02mbar.

[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. Calculated based on the carbon content of 50%, the amount of carbon dioxide fixed during the cultivation process is 5.99 kg.

[0088] Embodiment 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) Cultivating microalgae under high temperature and high light conditions, wherein the microalgae is Scenedesmus, and the conditions for culturing the microalgae are: an algae liquid temperature of 33.5° C., an algae liquid pH value of 7.1, and a light intensity of 92 μmol·m -2 ·s -1 , CO in the gas introduced 2 The concentration was 5%, and the microalgae were subcultured after entering the stable phase, and the subculture was performed 4 times;

[0091] Then, the CRISPR-Cas9 gene editing technology is used to improve the gene of the microalgae cytochrome P450 enzyme. The gene improvement method is to insert an enhancer into the gene promoter sequence of the cytochrome P450 enzyme to increase the expression level of the cytochrome P450 enzyme to 4.50 times the original level, thereby obtaining improved microalgae;

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

[0093] (3) inoculating the microalgae species acclimated in step (2) into the pretreated organic wastewater, wherein the pretreatment of the organic wastewater is to expose the organic wastewater to light for 24 hours, during which air is continuously introduced; after the microalgae species are inoculated into the organic wastewater, the optical density at a wavelength of 680 nm is 1.0, and the microalgae species are cultured in a horizontal tube photobioreactor, and the culture conditions of the microalgae are: temperature of 32.5° C., pH of 6.6, and light intensity of 93 μmol·m -2 ·s -1 , CO in the gas introduced 2 The concentration is 5.5%; during the culture process, the gene is expressed to generate cytochrome P450 enzyme, which accelerates the hydroxylation of organic pollutants and degrades organic pollutants in organic wastewater. The culture cycle of the microalgae is 6.5 days until the wastewater treatment is completed; the microalgae is harvested once after each culture cycle, and the method for harvesting the microalgae is centrifugal freeze-drying, the centrifugal speed is 5000rrpm, the freeze-drying temperature is -50°C, and the pressure is 0.1mbar.

[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. Calculated based on the carbon content of 50%, the amount of carbon dioxide fixed during the cultivation process is 4.22 kg.

[0095] Based on the above embodiments, it can be concluded that the method of the present invention improves the microalgae at the genetic level, and through the stimulation and regulation of high temperature and high light culture conditions, the expression of cytochrome P450 enzymes is enhanced, the hydroxylation of organic pollutants is promoted, the polarity is increased, and the organic pollutants are more easily degraded, thereby achieving the effect of treating organic wastewater, and at the same time, carbon dioxide can be fixed to obtain microalgae biomass; the method can treat various types of organic wastewater with high treatment efficiency and low cost.

[0096] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the method of the present invention, addition 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 comprises the following steps: (1) Cultivating microalgae under high temperature and high light conditions, and then using gene editing technology to improve the gene of cytochrome P450 enzyme of the microalgae, thereby increasing the expression level of cytochrome P450 enzyme and obtaining improved microalgae; (2) culturing the modified microalgae obtained in step (1) in a gradient manner in organic wastewater to obtain domesticated microalgae species; (3) inoculating the microalgae species domesticated in step (2) into the pretreated organic wastewater and culturing them in a photobioreactor, wherein the organic matter in the organic wastewater is degraded during the culturing process until the wastewater treatment is completed.

2. The method according to claim 1, characterized in that The microalgae in step (1) include Scenedesmus or Chlorella; Preferably, the conditions for culturing the microalgae in step (1) are: the algae solution temperature is 32-35°C, the algae solution pH is 6.5-7.1, and the light intensity is 90-110 μmol·m -2 ·s -1 , the CO2 concentration in the introduced gas is 5-10%; Preferably, during the culturing of the microalgae in step (1), the microalgae are subcultured after entering the stable phase, and the number of subcultures is more than 3 times; Preferably, the container used for the microalgae culture in step (1) includes a conical flask or a fermentation bottle.

3. The method according to claim 1 or 2, characterized in that: The gene editing technology in step (1) is CRISPR-Cas9 gene editing technology; Preferably, the gene modification in step (1) is performed by inserting an enhancer into the gene promoter sequence of the cytochrome P450 enzyme to increase the expression level of the cytochrome P450 enzyme; Preferably, the expression level of the cytochrome P450 enzyme is upregulated to 3.45 to 5.78 times of the original level.

4. The method according to any one of claims 1 to 3, characterized in that: Step (2) culturing the domesticated and improved microalgae in organic wastewater of different concentrations, wherein the organic wastewater is added with nutrients required for the growth of the microalgae; Preferably, 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.

5. The method according to claim 4, characterized in that The source of the organic wastewater to be treated includes any one or a combination of at least two of the cooling tower wastewater of a biomass power plant, the cooling tower wastewater of a coal-fired power plant, or the cooling tower wastewater of a chemical plant; Preferably, the concentration of benzotriazole in the organic wastewater to be treated is 6-10 mg / L.

6. The method according to any one of claims 1 to 5, characterized in that: The acclimatization culture in step (2) is carried out on a plant culture rack in a constant temperature greenhouse, and the culture container used includes a conical flask or a fermentation bottle; Preferably, the conditions for the acclimation culture in step (2) are the same as the culture conditions in step (1); Preferably, in step (2), microalgae species are domesticated and cultured that can grow in 100% concentration of the organic wastewater to be treated.

7. The method according to any one of claims 1 to 6, characterized in that: The pretreatment of the organic wastewater in step (3) is to expose the organic wastewater to light for more than 24 hours and continuously introduce air; Preferably, the optical density of the microalgae species after inoculation into the organic wastewater in step (3) is 0.8 to 1; Preferably, the photobioreactor in step (3) comprises any one of a raceway pool, a column-type photobioreactor or a horizontal tube-type photobioreactor.

8. The method according to any one of claims 1 to 7, characterized in that: The culture conditions of the microalgae in step (3) are: temperature of 32-35°C, pH of 6-7, and light intensity of 90-110 μmol·m -2 ·s -1 , the CO2 concentration in the introduced gas is 5-10%; Preferably, in step (3), nutrients are added to the photobioreactor during the microalgae cultivation process; Preferably, during the microalgae cultivation process in step (3), gene expression generates cytochrome P450 enzymes, which accelerate the hydroxylation of organic pollutants and thus promote the degradation of organic pollutants; Preferably, the culture period of the microalgae in step (3) is 5 to 8 days.

9. The method according to any one of claims 1 to 8, characterized in that: Step (3) harvesting the microalgae once every cultivation cycle; Preferably, the method for harvesting microalgae includes a centrifugal freeze-drying method, which is divided into two steps: centrifugation and freeze-drying; Preferably, the centrifugal speed is 5000-7000 rpm; Preferably, the freeze-drying temperature is -50 to -30°C, and the freeze-drying pressure is 0.02 to 0.1 mbar.

10. The method according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: (1) Cultivating microalgae under high temperature and high light conditions, wherein the microalgae include Scenedesmus or Chlorella, and the conditions for culturing the microalgae are: an algae liquid temperature of 32 to 35° C., an algae liquid pH value of 6.5 to 7.1, and a light intensity of 90 to 110 μmol·m -2 ·s -1 The CO2 concentration in the introduced gas is 5-10%, and the microalgae are subcultured after entering the stable period, and the number of subcultures is more than 3 times; Then, the CRISPR-Cas9 gene editing technology is used to improve the gene of the microalgae cytochrome P450 enzyme, wherein the gene improvement method is to insert an enhancer into the gene promoter sequence of the cytochrome P450 enzyme to increase the expression level of the cytochrome P450 enzyme to 3.45 to 5.78 times of the original, thereby obtaining improved microalgae; (2) The modified microalgae obtained in step (1) are subjected to gradient acclimation and culture in organic wastewater of different concentrations, wherein the concentration of the organic wastewater is 50-100% of the concentration of the organic wastewater to be treated, and the gradient is selected within the concentration range. The source of the organic wastewater to be treated includes any one or a combination of at least two of the cooling tower wastewater of a biomass power plant, the cooling tower wastewater of a coal-fired power plant, or the cooling tower wastewater of a chemical plant. The concentration of benzotriazole in the organic wastewater to be treated is 6-10 mg / L. The conditions of the acclimation and culture are the same as the high temperature and high light culture conditions in step (1), so as to obtain acclimated microalgae species capable of growing in 100% concentration of the organic wastewater to be treated; (3) inoculating the microalgae species acclimated in step (2) into pretreated organic wastewater, wherein the pretreatment of the organic wastewater is to expose the organic wastewater to light for more than 24 hours and continuously introduce air; the optical density of the microalgae species after inoculation into the organic wastewater is 0.8 to 1, and culturing the microalgae species in a photobioreactor, wherein the photobioreactor comprises any one of a raceway pool, a column-type photobioreactor, or a horizontal tube-type photobioreactor, and the culturing conditions of the microalgae are: a temperature of 32 to 35° C., a pH of 6 to 7, and a light intensity of 90 to 110 μmol·m -2 ·s -1 , the CO2 concentration in the introduced gas is 5-10%; during the cultivation process, gene expression generates cytochrome P450 enzyme, 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 method for harvesting the microalgae includes centrifugal freeze-drying, which is divided into two steps of centrifugation and freeze-drying, the centrifugal speed is 5000-7000rrpm, the freeze-drying temperature is -50--30°C, and the pressure is 0.02-0.1mbar.