Method for intensifying phycomycete symbiotic system by mutagenizing energy microalgae through plasma

Abstract

The invention provides a method for intensifying an algae-bacterium symbiotic system by utilizing plasma mutagenesis of energy microalgae, which comprises the following steps: step 1, mutagenizing microalgae in a logarithmic phase for different times by utilizing plasma to obtain mutagenized algae liquid corresponding to different times; step 2, screening the mutated algae liquid corresponding to different time to obtain a high-grease mutant algae strain; step 3, performing sterile sewage culture on the obtained high-grease mutant microalgae strain to obtain oil-producing mutant microalgae with purification and decontamination functions; step 4, determining unsterilized sewage with the optimal dilution concentration suitable for the growth of the oil-producing mutation microalgae; step 5, determining a strain beneficial to the growth of the oil-producing mutation microalgae in the unsterilized sewage with the optimal dilution concentration; step 6, carrying out mixed culture on the obtained strain and the oil-producing mutation microalgae to obtain a phycomycete symbiotic system; according to the invention, the biomass and grease accumulation of microalgae can be improved, and pollutants in sewage can be efficiently removed at the same time.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for strengthening an algae-bacteria symbiotic system by utilizing plasma to induce energy microalgae. Background Art

[0002] Since the increase in sewage production has been proven to be one of the most serious environmental problems conceived by humans, sewage contains organic components, suspended matter, and pollutants such as nitrogen and phosphorus. If it is directly discharged, it will pollute the local environment, increase the pollution load, and cause eutrophication. It is crucial to treat sewage reasonably, economically and effectively. Using sewage to cultivate microalgae can not only purify sewage, but also obtain raw materials for preparing bioenergy, which has many benefits, because the organic components, nitrogen and phosphorus in sewage can be absorbed and utilized by microalgae, promote their own growth, and increase lipid content, thereby achieving wastewater purification and resource utilization, greatly improving the feasibility of microalgae cultivation in wastewater. However, under natural conditions, microalgae have poor environmental tolerance and are easily affected by the characteristics of the wastewater itself, resulting in low growth efficiency. This is also the reason why microalgae purification technology is not commonly used in current wastewater treatment.

[0003] At present, mutation breeding technology is expected to change the poor tolerance of microalgae and the feasibility of effectively accumulating biomass in wastewater. Plasma (ARTP) mutagenesis is a new artificial mutagenesis technology. Compared with other mutagenesis methods, the active particles produced by ionized gas have strong penetration and higher mutagenesis efficiency. It is a breeding technology with broad application prospects. At present, studies have shown that the biomass accumulation of high-lipid algae strains induced by ARTP is much higher than that of natural algae strains during the cultivation process, and at the same time, it has stronger tolerance and efficient lipid production in sewage culture.

[0004] In addition, the microbial community in sewage is complex. There are not only bacteria that can increase the growth of algae and the oil content, but also bacteria that inhibit the growth of microalgae. The selected chain algae is a microalgae suitable for growth in sewage, with a strong ability to absorb nutrients such as nitrogen and phosphorus. However, the accumulation of algae biomass in the natural state is poor and usually does not meet the requirements of energy utilization. The use of artificial mutagenesis to transform microalgae to achieve highly tolerant and efficient oil production characteristics, while analyzing the mutant microalgae and oil accumulation and the interaction with bacteria in sewage has important scientific significance for establishing a stable algae-bacteria symbiotic system. Summary of the invention

[0005] The purpose of the present invention is to provide a method for strengthening the algae-bacteria symbiotic system by using plasma to induce energy microalgae, which solves the defects of mutant microalgae and oil accumulation and inability to coexist with bacteria in sewage.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: The present invention provides a method for strengthening the algae-bacteria symbiotic system by using plasma to induce energy microalgae, comprising the following steps: Step 1, using plasma to induce mutagenesis of microalgae in a logarithmic growth phase for different periods of time, to obtain induced algae liquid corresponding to different periods of time; Step 2, screening high-lipid mutant algae strains from the algae liquid after mutagenesis at different times; Step 3, culturing the obtained high-lipid mutant algae strain in sterile sewage to obtain oil-producing mutant microalgae with purification and decontamination capabilities; Step 4, determining the optimal dilution concentration of non-sterilized sewage suitable for the growth of oil-producing mutant microalgae; Step 5, determining the strains that are conducive to the growth of oil-producing mutant microalgae in the unsterilized sewage at the optimal dilution concentration; Step 6, mixing and culturing the obtained bacterial strain and the oil-producing mutant microalgae to obtain an algae-bacteria symbiotic system.

[0007] Preferably, in step 1, the microalgae in the logarithmic growth phase are mutated using plasma to obtain a mutagenized algae solution, and the specific method is: The algae liquid is placed in a plasma reactor, and the algae liquid is induced for different time periods with a set input voltage, current and air flow rate to obtain the induced algae liquid corresponding to different time periods.

[0008] Preferably, in step 2, high-lipid mutant algae strains are screened from the mutagenized algae liquids at different times, and the specific method is: The obtained mutagenic algae solutions corresponding to different times are respectively cultured in a sterile protective nutrient solution in the dark to obtain algae solutions after dark culture at different times; Spreading each dark-cultured algae liquid on a solid plate substrate for culture to obtain multiple algae colonies; The primary algae strains were obtained from the algae colonies with a mortality rate of more than 95%; The primary selected algae strains were rapidly cultured, and mutant algae strains with high oil yield were obtained through the three-index screening principle.

[0009] Preferably, the three-index screening principle is: The first screening principle is that the specific growth rate is 10% greater than that of the normal strain, and the relative fluorescence value is 20% greater than that of the normal strain; The second screening principle is that the product of the specific growth rate and the relative fluorescence value is greater than 35% of the control strain.

[0010] Preferably, in step 3, the obtained high-lipid mutant algae strain is cultured in sterile sewage to obtain oil-producing mutant microalgae with purification and decontamination capabilities, and the specific method is: The obtained high-lipid mutant algae strain is inverted and cultured in sterile sewage to obtain mutant microalgae with color and diameter that meet the requirements; The obtained mutant microalgae are rapidly cultured to obtain oil-producing mutant microalgae capable of purifying and removing pollutants.

[0011] Preferably, in step 4, the optimal dilution concentration of non-sterilized sewage suitable for the growth of oil-producing mutant microalgae is determined by: The obtained oil-producing mutant microalgae is subjected to sterile sewage shake flask acclimation culture to obtain acclimated mutant microalgae; The domesticated mutant microalgae are cultured in shake flasks in non-sterile sewage of different dilution concentrations to form an initial algae-bacteria symbiotic system; According to the biomass and oil accumulation of the domesticated mutant microalgae and the pollutant removal efficiency of the sewage in the initial algae-bacteria symbiotic system, the optimal dilution concentration of the non-sterilized sewage suitable for the growth of the domesticated mutant microalgae is determined.

[0012] Preferably, in step 5, the strains that are conducive to the growth of oil-producing mutant microalgae in the unsterilized sewage at the optimal dilution concentration are determined by: Determine the strain that is beneficial to the growth of mutant microalgae after domestication in non-sterilized sewage; Selecting dominant strains from the obtained strains by solid plate coating; The non-sterilized sewage of the best dilution concentration is coated on a solid plate, and cultured at a constant temperature to separate the dominant bacterial colonies and obtain the dominant bacterial colonies; The solid plate streaking method is used to purify the dominant bacterial colonies to obtain purified bacterial colonies; The purified bacterial colonies were activated and cultured in a constant temperature shaker to obtain dominant strains; The obtained dominant strains and dominant bacterial species are used as bacterial species that are conducive to the growth of oil-producing mutant microalgae.

[0013] Preferably, high-throughput sequencing technology is used to determine the strains that are beneficial to the growth of the mutant microalgae after domestication in non-sterilized sewage.

[0014] Preferably, in step 1, the microalgae in the logarithmic growth phase are mutated using plasma to obtain a mutagenized algae solution, and the specific method is: The algae liquid is placed in a plasma reactor, and the algae liquid is induced for different time periods with a set input voltage, current and air flow rate to obtain the induced algae liquid corresponding to different time periods; In step 2, high-lipid mutant algae strains are screened from the mutagenized algae liquids at different times, and the specific method is: The obtained mutagenic algae solutions corresponding to different times are respectively cultured in a sterile protective nutrient solution in the dark to obtain algae solutions after dark culture at different times; Spreading each dark-cultured algae liquid on a solid plate substrate for culture to obtain multiple algae colonies; The primary algae strains were obtained from the algae colonies with a mortality rate of more than 95%; The primary selected algae strains are rapidly cultured, and mutant algae strains with high oil yield are obtained through the three-index screening principle.

[0015] An algae-bacteria symbiotic system is obtained by strengthening the method described.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for constructing a high-efficiency plasma-induced mutagenesis energy microalgae and bacterial symbiotic system. In the method, the microalgae are first subjected to ARTP mutagenesis to preliminarily screen out algae strains with high oil yield, and then screened and domesticated through sterilized sewage to obtain mutant energy algae strains with high biomass and oil accumulation and removal of high pollutants, thereby constructing a bacterial-algae symbiotic system of mutant energy microalgae and dominant bacteria in sewage.

[0017] In order to better reflect the ability of the induced energy microalgae to utilize pollutants in sewage and the potential of forming a stable algae-bacteria symbiotic system with bacteria in sewage to purify water bodies, the accumulation of biomass and oil content of the induced microalgae is taken into account. The present invention performs ARTP mutagenesis on microalgae, obtains algae strains with high oil yield through preliminary screening, and then screens out mutant energy microalgae suitable for growth in sewage in a solid plate that is a nutrient substance for sterilized sewage. Then, through sterilized sewage domestication, mutant algae strains with higher biomass and oil accumulation and removal of high pollutants are obtained, and the induced mutant energy microalgae are cultivated in sewage with dilution multiples of 2, 4, 6, 8, 10, and 20 times to construct a bacteria-algae symbiotic system. By observing the biomass and oil accumulation of mutant energy microalgae and the pollutant removal efficiency of sewage in the algae-bacteria symbiotic system, the sewage concentration most suitable for the growth of mutant energy microalgae is determined. The changes in the functional structure of the bacterial community in the optimal concentration of bacterial sewage are analyzed to obtain dominant strains. Then, the sewage culture solution is coated with a solid plate to separate the dominant bacteria in the sewage, and the separated dominant bacteria are mixed with mutant energy microalgae in sterilized sewage at different inoculation concentrations and different species combinations to further evaluate the operational stability of the symbiotic system, and continuously optimize the culture conditions including temperature, pH, and algae-bacteria inoculation ratio. Through the growth of mutant microalgae, oil accumulation, pollutant utilization efficiency, and changes in microbial communities, a stable and efficient bacteria-algae symbiotic system is established. To prove the advantages of microalgae induced by ARTP in sewage compared with the original microalgae. Whether the oil yield of microalgae can be increased and the cultivation cost can be reduced, the key is to screen out mutant energy microalgae that can reproduce rapidly and maintain a high oil content in sewage culture, and at the same time form a stable algae-bacteria symbiotic system with bacteria in sewage. Therefore, the method of constructing a stable algae-bacteria symbiotic system with mutant energy microalgae and bacteria in sewage plays an increasingly important role in the field of biomass energy and sewage purification. The efficient algae-bacteria symbiotic system constructed by the present invention can be used in the following aspects: (1) It is possible to cultivate mutant microalgae with strong tolerance and high oil production in sewage, which has important scientific significance for establishing a stable algae-bacteria symbiotic system; (2) It can improve the oil accumulation and pollutant utilization efficiency of mutant energy microalgae in sewage, and has great potential in the field of biomass energy and sewage recycling; (3) The migration and change patterns of the microbial community structure of the dominant bacterial species in the mutant energy microalgae and the sewage cultured microalgae can be obtained, and the interaction patterns of algae and fungi can be analyzed, which has great theoretical significance for the in-depth analysis of the algae-fungus symbiosis mechanism.

[0018] In summary, the present invention first obtains a high-oil-yielding mutant energy algae strain through ARTP mutagenesis, screens out high-oil-yielding mutant energy microalgae suitable for growing in sewage through sterilized sewage, and domesticates the high-oil-yielding mutant energy algae strain in sterilized sewage to obtain a mutant energy algae strain with high biomass and oil accumulation and high pollutant utilization efficiency. After domestication, the mutant energy microalgae is cultured in sewage diluted at different times to construct a bacteria-algae symbiotic system. The biomass and oil accumulation of the mutant energy microalgae, the pollutant utilization efficiency of the sewage, and the changes in the microbial community in the algae-bacteria symbiotic system are observed. Then, the sewage culture solution is solid-plated to separate the dominant bacteria in the sewage, and the separated dominant bacteria are mixed and cultured with the mutant energy microalgae in sterilized sewage in different combinations, and the operational stability of the symbiotic system is further evaluated, thereby establishing a stable and efficient bacteria-algae symbiotic system. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a comparison of the production capacity of high-oil mutant algae strains and original algae strains; Figure 2 It is the biomass and oil accumulation of microalgae in sterilized and non-sterilized sewage; Figure 3 It is the utilization efficiency of pollutants in sewage; Figure 4 It is the changing law of microbial communities in sewage. DETAILED DESCRIPTION

[0020] In the following description, specific details such as specific system structures, technologies, etc. are provided for the purpose of illustration rather than limitation, so as to provide a thorough understanding of the embodiments of the present application. However, it should be clear to those skilled in the art that the present application may also be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices, circuits, and methods are omitted to prevent unnecessary details from obstructing the description of the present application.

[0021] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or combinations thereof.

[0022] It should also be understood that the term “and / or” used in the specification and appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0023] As used in the specification and appended claims of this application, the term "if" can be interpreted as "when" or "uponce" or "in response to determining" or "in response to detecting", depending on the context. Similarly, the phrase "if it is determined" or "if [described condition or event] is detected" can be interpreted as meaning "uponce it is determined" or "in response to determining" or "uponce [described condition or event] is detected" or "in response to detecting [described condition or event]", depending on the context.

[0024] In addition, in the description of the present application specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the descriptions and cannot be understood as indicating or implying relative importance.

[0025] References to "one embodiment" or "some embodiments" etc. described in the specification of this application mean that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, the statements "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in different places in this specification do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[0026] Example 1 The present embodiment provides a method for strengthening the algae-bacteria symbiotic system by using plasma to induce energy microalgae. In the method, the microalgae are first induced by ARTP to preliminarily screen out algae strains with high oil production, and then screened and domesticated through sterilized sewage to obtain mutant energy algae strains with high biomass and oil accumulation and high pollutant removal, and to construct a bacteria-algae symbiotic system of mutant energy microalgae and dominant bacteria in sewage, which specifically includes the following steps: Step 1, subjecting microalgae in a logarithmic growth phase to mutagenesis of algae solution at different times on a plasma instrument with a set input voltage, current and air flow rate to obtain mutagenesis algae solution corresponding to different times; The obtained mutagenic algae solutions corresponding to different times are transferred to sterile protective nutrient solutions respectively, and dark culture is performed to prevent the mutant microalgae from recovering their properties due to light repair, thereby obtaining dark cultured algae solutions; The obtained dark cultured algae liquid is respectively applied on a solid plate substrate for culture to obtain a plurality of algae colonies; The obtained multiple algae colonies are compared with the non-mutated microalgae, wherein the primary algae strains are obtained from the algae colonies with a lethality rate of more than 95%; The obtained preliminary selected algae strains were selected into 6-well plates for rapid culture, and the mutant algae strains with high oil yield were obtained by comparing the specific growth rate, relative fluorescence value and the product value of the specific growth rate and relative fluorescence value of the preliminary selected algae strains in the 6-well plates; The above process is the mutagenesis breeding step set in the present application, which can realize the rapid screening of high-oil-yielding microalgae in a short time, wherein the mutagenesis is carried out by atmospheric pressure and room temperature plasma mutagenesis, and the plasma is used to make the microalgae cells produce a high fault-tolerant repair process, thereby producing a variety of random traits; dark culture is to protect the various random traits from disappearing or recovering under the photosynthetic repair effect of the microalgae under light conditions; the purpose of plate culture is mainly to test the growth of the induced algae strain through solid culture medium, and the influence of the mutagenesis parameters on the microalgae, such as the lethal effect, can be quickly and accurately determined; the main purpose of 6-well plate culture is to quickly carry out liquid culture of the selected target algae colonies, and physiological index tests can be carried out in a relatively short time to realize the identification of mutant traits of oil-producing algae strains.

[0027] Step 2, evenly spreading the selected high-oil-yielding mutant algae strains on a solid plate substrate with sterile sewage as a nutrient, and inverting the plate substrate in a light incubator to observe the growth of the microalgae on the culture medium; and selecting the mutant microalgae with color and diameter that meet the requirements; The selected mutant microalgae are cultivated in batches using a 6-well plate rapid culture method to obtain oil-producing mutant microalgae; The obtained oil-producing mutant microalgae is acclimated and cultured in a sterile sewage shake flask to obtain acclimated mutant microalgae.

[0028] The above cultivation is the process of screening superior oil-producing algae strains that can quickly absorb nutrients in sewage by sewage solid plate cultivation. Among them, sterile sewage cultivation is to conduct organic and inorganic mixed nutrient acclimation test on the oil-producing algae strains obtained in step 2. If algae colonies can be grown on the sewage culture medium solid plate, it means that they have the characteristics of purification and pollution removal. The color and size determine the efficiency of the oil-producing algae strains in utilizing nutrients in sterile sewage. The purpose of the 6-well plate culture here is to quickly obtain a high-density number of target algae strains (the culture medium used here is conventional BG-11 culture medium); finally, the obtained high-density algae strains are acclimated to sterile sewage, and this purpose can be achieved by means of sterile sewage shaking bottles.

[0029] Step 3, the domesticated mutant microalgae are cultured in shake flasks in non-sterile sewage of different dilution concentrations to form an initial algae-bacteria symbiotic system, and the domesticated mutant microalgae are cultured in sterilized sewage and raw sewage as a control group. The biomass and oil accumulation of the domesticated mutant microalgae in the initial algae-bacteria symbiotic system corresponding to the non-sterile sewage, and the pollutant removal efficiency of the sewage are observed to determine the optimal non-sterile sewage dilution concentration suitable for the growth of the domesticated mutant microalgae; High-throughput sequencing technology was used to analyze the changes in the functional structure of the bacterial community in non-sterilized sewage to determine the best strain for the growth of mutant microalgae after domestication; The dominant strains were selected from the obtained strains by solid plate spreading.

[0030] Step 4, using a plate coating method, coating the unsterilized sewage at the optimal dilution concentration on a solid plate, and performing constant temperature culture to separate the dominant bacterial colonies to obtain the dominant bacterial colonies; The dominant colonies are purified by solid plate streaking method until only one colony morphology can be observed to obtain purified colonies; The purified colonies were activated and cultured on a constant temperature shaker to obtain dominant bacterial strains, which were then identified to verify whether the dominant strain obtained in step 3 was of the same species as the dominant bacterial strain.

[0031] If the obtained dominant strain and the separated dominant bacterial species are of the same genus, the dominant strain or dominant bacterial species is mixed with the domesticated mutant microalgae obtained in step 2 in sterile sewage to obtain an algae-bacteria symbiotic system; If the obtained dominant strains and the separated dominant bacterial species are of multiple genera, the dominant strains and dominant bacterial species are mixed and cultured in different (species, concentration) combinations with the domesticated mutant microalgae obtained in step 2 in sterile sewage to obtain an algae-bacteria symbiotic system; By regulating the operating conditions of the algae-bacteria symbiosis system, the production capacity and wastewater utilization of the algae-bacteria symbiosis system are optimized. The growth morphology of microalgae and the changes in the algae-bacteria symbiosis system, as well as the changes in algae-bacteria biomass, microalgae photosynthesis efficiency, oil composition and physiological characteristics of algae and bacteria are monitored to further evaluate the operating stability of the symbiotic system.

[0032] This application uses plasma mutagenesis to obtain high-oil-producing mutant algae strains, and then screens the superior oil-producing microalgae with pollution resistance and efficient utilization of pollutants in sewage through sterilized sewage cultivation. This is a major problem that needs to be overcome in sewage cultivation of oil-producing microalgae; and to achieve mixed and efficient cultivation of sewage, it is necessary to overcome the interference of the bacterial environment. The oil-producing microalgae enhanced by plasma mutagenesis have environmental tolerance, including antibacterial properties. The mixed cultivation process of bacterial sewage is a process of mutual influence between the superior oil-producing microalgae and the bacterial community. With the favorable growth of microalgae, an algae-bacteria symbiotic system is gradually formed. By regulating the system conditions, the efficient operation of the algae-bacteria symbiotic system can be enhanced, which is an important control measure for forming a stable system. At the same time, it also provides a theoretical and research basis for directly adding functional microalgae to sewage in the future to promote the formation of a new symbiotic algae-bacteria system.

[0033] Example 2 This embodiment provides a method for constructing an efficient plasma-induced energy microalgae and bacterial symbiotic system, which uses ARTP-induced high-oil-yielding mutant microalgae and dominant bacteria in sewage to construct an algae-bacteria symbiosis. The algae liquid is first induced by ARTP to produce high-oil-yielding mutant microalgae, then screened and domesticated with sterilized sewage, and cultured in sewage to obtain dominant bacteria. Finally, the mutant microalgae and the dominant bacteria are mixed and cultured to obtain an efficient and stable bacteria-algae symbiotic system. Specifically, it includes the following steps: Step 1: Mutate the microalgae. 680 The absorbance is 0.4-0.6, at which time the microalgae is in the logarithmic growth phase, the cells are more active and more prone to mutation.

[0034] The power supply input voltage is controlled to be 80V, the current is 1A, and the air flow rate is 5L / min. In this example, the mutagenesis time is adjusted as an example. The mutagenesis radiation time of the algae solution is 0 s, 10 s, 20 s, 30 s, 40 s, 50 s, 60 s, 65 s, and 70 s. Set the power supply input voltage to 30 V and the current to 2 A to start the instrument, and after preheating for 10 min, adjust the power supply voltage and frequency knob to the input voltage of 80V and the current of 1A to ensure the stable operation of the instrument. At this time, the actual discharge power of the plasma is about 42.90W. Keep the power supply frequency knob unchanged, return the voltage knob to zero, turn off the power switch, take 200ul of algae solution to the sterilized centrifuge tube cap and put it into the DBD plasma reactor, turn on the power switch again, and adjust the voltage knob to 80V to start mutagenesis. The algae solution is mutated by adjusting different mutagenesis times to obtain the mutagenesis algae solution corresponding to different mutagenesis times.

[0035] The sterile protective nutrient solution is a mixture of 5 mL of culture medium and 5 mL of 10% glycerol. The 100 μL algae solution after mutation is transferred to the protective nutrient solution together with the sterilized lid and cultured in the dark for 2-3 hours. Take 200 μL of the mutagenic algae solution and add it to a 90 mm solid plate containing the protective nutrient solution medium and evenly spread it with a sterilized triangular coating rod, invert it and place it in a constant temperature and light incubator for culture. Spread three plates as parallels, and use the unmutated algae solution as a blank control. Both solid culture and subsequent 6-well plate culture require culture in a constant temperature and light incubator, with a light cycle of 12 h: 12 h and a culture temperature of 25±1℃. After 10 days of solid culture, the algae colonies of the mutagenic group and the control group are counted, and the lethality rate of more than 95% is used as the preliminary screening index for the optimal mutagenic time. Select mutant algae strains with larger diameters and darker colors to culture quickly in 6-well plates. The 6-well plate culture method can achieve rapid culture of mutant algae and shorten the entire mutagenesis time. The first screening principle for screening out high-efficiency production-yielding microalgae is that the specific growth rate is greater than 10% of the normal strain, and the relative fluorescence value is greater than 20% of the normal strain; the second screening principle is that the product of the specific growth rate and the relative fluorescence value is greater than 35% of the control strain, and high specific growth rate mutants or high relative fluorescence mutants are screened for leaks.

[0036] Step 2: sterilize the sewage, and spread the mutant energy algae strain on a solid plate in the sterilized sewage, using the pollutants in the sewage as nutrients to promote the growth of mutant energy microalgae, thereby screening out mutant microalgae that are more suitable for survival in the sewage environment. The experiment was placed in a constant temperature and light incubator for cultivation, and the culture conditions were a temperature of 25±1°C and a light intensity of 80 μmol·m -2 ·s -1 The light / dark cycle was 12:12. The nutrient solution in the 6-well plate for rapid culture was sterilized sewage. The absorbance on the 3rd and 8th days was recorded. After 10 days, the mutant energy microalgae in the 6-well plate were transferred to the shaking bottle for domestication. The domestication period was 8 days. The domestication effect reached the microalgae biomass accumulation of >180mg / L, the oil content of >150mg / L, and the TOC, COD, and NH 4 + The degradation effects of -N, TN, and TP were above 25%, 20%, 70%, 60%, and 90%. The acclimatization culture conditions were the same as above. The initial pH of the sterilized wastewater was set to 7.0 ± 0.1 before culture. The pH value fluctuated during the culture process and no additional adjustment was required.

[0037] Step 3, culture the domesticated mutant energy microalgae in non-sterilized sewage of different dilution concentrations, with dilution multiples of 2, 4, 6, 8, 10, and 20 times, with raw sewage and sterilized sewage as the control group. By constructing a bacterial-algae symbiotic system in sewage of different dilution concentrations, observe the growth, oil accumulation, and pollutant utilization efficiency of the mutant microalgae to determine the sewage concentration most suitable for the growth of the mutant microalgae. The optimal sewage culture effect achieves microalgae biomass accumulation> 250mg / L, oil content> 200mg / L, TOC, COD, NH 4 + The degradation effects of -N, TN, and TP were above 35%, 30%, 80%, 75%, and 95%, respectively. The dominant strains were then obtained by analyzing the changes in the functional structure of the flora in the best bacterial sewage. The pollutants in the sewage were measured according to the fourth edition of the "Water and Wastewater Monitoring and Analysis Methods". At the same time, high-throughput sequencing technology was used to observe the changes in the function and structure of the microbial community.

[0038] Step 4, using the plate coating method, the best sewage culture solution is solid plate coated, and constant temperature culture is performed to separate the dominant colonies; the dominant bacteria are purified by the solid plate streaking method until only one colony morphology can be observed. The purification and culture of the dominant bacteria is carried out in sterilized sewage, and the strain identification is analyzed by ITS evolutionary tree. The purified and identified dominant bacteria are cultured in sterilized sewage with different inoculation concentrations and different types of combinations with mutant energy microalgae to form an algae-bacteria symbiotic system, and the culture conditions including temperature, pH, and algae-bacteria inoculation ratio are continuously optimized, through the growth of mutant microalgae, oil accumulation and pollutant utilization efficiency and changes in microbial communities. The growth morphology of the microalgae in the culture is observed by a high-power microscope and a scanning electron microscope to observe the changes in the algae-bacteria symbiotic system, to construct an efficient and stable bacteria-algae symbiotic system, and finally establish an efficient algae-bacteria symbiotic system suitable for mixed cultivation of mutant microalgae sewage.

[0039] Implementation, 3 This example uses Scenedesmus as an example to explain this method in detail. The Scenedesmus in this example was purchased from the freshwater algae seed bank of Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. The ARTP mutagenesis system used was purchased from Nanjing Suman Plasma Technology Co., Ltd.

[0040] Step 1. Cultivate Scenedesmus using BG11 medium. Inoculate 200 mL of Scenedesmus algae solution into a 500 mL conical flask. Seal the flask with a sterile film and place it in a constant temperature and light incubator. Cultivate in semi-continuous culture mode. Average light intensity: 80 μmol·m -2 ·s -1 , photoperiod: 12L:12D, culture temperature: (25±1)°C. The positions of the conical flasks were randomly changed and shaken 3-4 times every day.

[0041] Step 2. When the culture reaches the logarithmic phase, the mutagenesis was performed. The mutagenesis instrument controlled the current to 1 A, the air flow rate to 5 L / min, the mutagenesis distance to 2 mm, and preheated for 10 min. The mutagenesis radiation voltage of the algae solution was adjusted to 120 V and the mutagenesis radiation time to 65 s.

[0042] Step 3. Transfer the induced algae solution to the sterile protective nutrient solution prepared in advance (5 mL BG11 medium + 5 mL 10% glycerol solution) and culture in the dark for 2-3 hours; Step 4. Take 1 mL of the mutagenized algae solution and dilute it 10 times in the protective nutrient solution. Take 200 μL of the diluted algae solution and add it to a 90 mm solid plate containing the protective nutrient solution medium and spread it evenly with a sterilized triangular coating rod. Immediately cover the lid and invert it into a constant temperature and light incubator for culture. Spread three plates under each mutagenesis condition as parallels, and use the unmutated algae solution as a blank control. Both solid culture and subsequent 6-well plate culture are required to be cultured in a constant temperature and light incubator with a light cycle of 12 h: 12 h and a culture temperature of 25±1°C. After 10 days of solid culture, count the algae colonies in the mutagenesis group and the control group, calculate the mortality rate and draw a mortality curve. The mortality rate is calculated according to the following formula: Lethality rate = (number of algae in the non-mutated group - number of algae in the mutated group) / number of algae in the non-mutated group × 100% The optimal mutagenesis time was determined by taking a lethality of 95% or more. The mutant algae strains with larger diameter and darker color were selected with a sterilized inoculation loop and quickly cultured in a 6-well plate.

[0043] Step 5. The mutants with high oil yield were obtained by comparing the growth rate, relative fluorescence value and the product of the specific growth rate and relative fluorescence value of the microalgae in the 6-well plate as screening indicators; the specific growth rate and relative fluorescence value were determined by the absorbance value measured at 680 nm by an ultraviolet spectrophotometer and the fluorescence value at an emission wavelength of 570 nm by staining the mutant algae solution with Nile red under a fluorescence spectrophotometer (calculated and determined by the excitation wavelength of 480 and the emission wavelength range of 500-650 nm). The first screening principle was that the specific growth rate was greater than 10% of the normal strain and the relative fluorescence value was greater than 20% of the normal strain; the second screening principle was that the product of the specific growth rate and the relative fluorescence value was greater than 35% of the control strain, and the mutants with high specific growth rate or high relative fluorescence value were screened for leak-filling.

[0044] Step 6. Sterilize the wastewater first, evenly spread the selected high-oil-yielding mutant algae strains on a solid culture medium with sterile wastewater as the nutrient and invert the culture in a light incubator to observe the growth of the microalgae on the culture medium; the experiment was carried out in a constant temperature light incubator with a culture temperature of 25±1°C and a light intensity of 80μmol m−2 ·s −1 , the light / dark cycle was 12:12.

[0045] Step 7. The selected algae colonies were cultured in bacterial wastewater using the 6-well plate rapid culture method. The wastewater in the 6-well plate was pretreated in different ways: (1) the wastewater was pretreated by sedimentation for 2 h; (2) the wastewater was pretreated by filtration through 0.45 μm and 0.22 μm membranes. The unpretreated wastewater was used as the control group. The biomass and lipid accumulation of the mutant microalgae in the well plate were observed to determine the most suitable wastewater pretreatment method for the growth of mutant energy microalgae. The biomass and lipid content were measured by the absorbance value at 680 nm using an ultraviolet spectrophotometer and the fluorescence value at an emission wavelength of 570 nm using a fluorescence spectrophotometer after staining the mutant algae liquid with Nile red.

[0046] Step 8. The mutant energy microalgae were cultured in shake flasks in the best pretreated sewage to form an algae-bacteria symbiotic system, and the sterilized sewage culture was used as the control group. The culture conditions were a temperature of 25±1°C and a light intensity of 80μmol m −2 ·s −1 , the light / dark cycle was 12:12, and the initial pH of the wastewater was set to 7.0±0.1 before cultivation. The pH value fluctuated during the cultivation process and no additional adjustment was required. The biomass and oil accumulation of mutant energy microalgae, the pollutant removal efficiency of sewage, and the changes in microbial communities in the algae-bacteria symbiotic system were observed. The biomass and oil content were measured by the absorbance value at 680nm using an ultraviolet spectrophotometer and the fluorescence value at an emission wavelength of 570nm using a fluorescence spectrophotometer by staining the mutant algae liquid with Nile red. Pollutants in sewage were measured according to the fourth edition of the "Water and Wastewater Monitoring and Analysis Methods".

[0047] By combining the energy microalgae induced by plasma with the bacterial symbiotic system, the accumulated biomass and oil of the mutant microalgae in this efficient algae-bacteria symbiotic system reached more than 354 mg / L and more than 220 mg / L, respectively, which were 54% and 20% higher than the normal strains; at the same time, the utilization rates of total nitrogen (TN), total phosphorus (TP) and total organic carbon (TOC) in sewage exceeded 87%, 99% and 47% respectively; it has great potential in the field of biomass energy and sewage recycling. In terms of biological communities, the community diversity of the mutant energy microalgae sewage culture system remained at a low level and the community structure was relatively stable throughout the culture process. The function of its microbial community was most significant in the metabolic process, especially the metabolism of carbohydrates, amino acids and growth factors, which further explained the interaction between microorganisms and the high consumption of substances in sewage, which was conducive to the realization of algae-bacteria symbiotic culture.

[0048] Example 4 This embodiment provides an algae-bacteria symbiotic system, which is obtained by strengthening the method described in Example 1.

[0049] The embodiments described above are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application, and should all be included in the protection scope of the present application.

Claims

1. A method for strengthening algae-bacteria symbiotic system by using plasma to induce energy microalgae, characterized in that: The following steps are involved: Step 1, using plasma to induce mutagenesis of microalgae in a logarithmic growth phase for different periods of time, to obtain induced algae liquid corresponding to different periods of time; Step 2, screening high-lipid mutant algae strains from the algae liquid after mutagenesis at different times; Step 3, culturing the obtained high-lipid mutant algae strain in sterile sewage to obtain oil-producing mutant microalgae with purification and decontamination capabilities; Step 4, determining the optimal dilution concentration of non-sterilized sewage suitable for the growth of oil-producing mutant microalgae; Step 5, determining the strains that are conducive to the growth of oil-producing mutant microalgae in the unsterilized sewage at the optimal dilution concentration; Step 6, mixing and culturing the obtained bacterial strain and the oil-producing mutant microalgae to obtain an algae-bacteria symbiotic system.

2. The method of strengthening algae-bacteria symbiotic system by using plasma to induce energy microalgae according to claim 1, characterized in that: Step 1, using plasma to induce mutagenesis of microalgae in the logarithmic growth phase to obtain an induced algae solution, the specific method is: The algae liquid is placed in a plasma reactor, and the algae liquid is induced for different time periods with a set input voltage, current and air flow rate to obtain the induced algae liquid corresponding to different time periods.

3. The method of strengthening algae-bacteria symbiotic system by using plasma to induce energy microalgae according to claim 1, characterized in that: In step 2, high-lipid mutant algae strains are screened from the mutagenized algae liquids at different times, and the specific method is: The obtained mutagenic algae solutions corresponding to different times are respectively cultured in a sterile protective nutrient solution in the dark to obtain algae solutions after dark culture at different times; Spreading each dark-cultured algae liquid on a solid plate substrate for culture to obtain multiple algae colonies; The primary algae strains were obtained from the algae colonies with a mortality rate of more than 95%; The primary selected algae strains were rapidly cultured, and mutant algae strains with high oil yield were obtained through the three-index screening principle.

4. The method of strengthening algae-bacteria symbiotic system by using plasma to induce energy microalgae according to claim 3, characterized in that: The three-index screening principle is as follows: The first screening principle is that the specific growth rate is 10% greater than that of the normal strain, and the relative fluorescence value is 20% greater than that of the normal strain; The second screening principle is that the product of the specific growth rate and the relative fluorescence value is greater than 35% of the control strain.

5. The method of strengthening algae-bacteria symbiotic system by using plasma to induce energy microalgae according to claim 1, characterized in that: In step 3, the obtained high-lipid mutant algae strain is cultured in sterile sewage to obtain oil-producing mutant microalgae with purification and decontamination capabilities, and the specific method is: The obtained high-lipid mutant algae strain is inverted and cultured in sterile sewage to obtain mutant microalgae with color and diameter that meet the requirements; The obtained mutant microalgae are rapidly cultured to obtain oil-producing mutant microalgae capable of purifying and removing pollutants.

6. The method of strengthening algae-bacteria symbiotic system by using plasma to induce energy microalgae according to claim 1, characterized in that: In step 4, the optimal dilution concentration of non-sterilized sewage suitable for the growth of oil-producing mutant microalgae is determined by: The obtained oil-producing mutant microalgae is subjected to sterile sewage shake flask acclimation culture to obtain acclimated mutant microalgae; The domesticated mutant microalgae are cultured in shake flasks in non-sterile sewage of different dilution concentrations to form an initial algae-bacteria symbiotic system; According to the biomass and oil accumulation of the domesticated mutant microalgae and the pollutant removal efficiency of the sewage in the initial algae-bacteria symbiotic system, the optimal dilution concentration of the non-sterilized sewage suitable for the growth of the domesticated mutant microalgae is determined.

7. The method of strengthening algae-bacteria symbiotic system by using plasma to induce energy microalgae according to claim 1, characterized in that: In step 5, the optimal dilution concentration of the bacterial species that is conducive to the growth of oil-producing mutant microalgae in the unsterilized sewage is determined by: Determine the strain that is beneficial to the growth of mutant microalgae after domestication in non-sterilized sewage; Selecting dominant strains from the obtained strains by solid plate coating; The non-sterilized sewage of the best dilution concentration is coated on a solid plate, and cultured at a constant temperature to separate the dominant bacterial colonies and obtain the dominant bacterial colonies; The solid plate streaking method is used to purify the dominant bacterial colonies to obtain purified bacterial colonies; The purified bacterial colonies were activated and cultured in a constant temperature shaker to obtain dominant strains; The obtained dominant strains and dominant bacterial species are used as bacterial species that are conducive to the growth of oil-producing mutant microalgae.

8. The method of strengthening algae-bacteria symbiotic system by using plasma to induce energy microalgae according to claim 7, characterized in that: High-throughput sequencing technology was used to identify strains in non-sterilized sewage that were beneficial to the growth of domesticated mutant microalgae.

9. The method of strengthening algae-bacteria symbiotic system by using plasma to induce energy microalgae according to claim 1, characterized in that: Step 1, using plasma to induce mutagenesis of microalgae in the logarithmic growth phase to obtain an induced algae solution, the specific method is: The algae liquid is placed in a plasma reactor, and the algae liquid is induced for different time periods with a set input voltage, current and air flow rate to obtain the induced algae liquid corresponding to different time periods; In step 2, high-lipid mutant algae strains are screened from the mutagenized algae liquids at different times, and the specific method is: The obtained mutagenic algae solutions corresponding to different times are respectively cultured in a sterile protective nutrient solution in the dark to obtain algae solutions after dark culture at different times; Spreading each dark-cultured algae liquid on a solid plate substrate for culture to obtain multiple algae colonies; The primary algae strains were obtained from the algae colonies with a mortality rate of more than 95%; The primary selected algae strains were rapidly cultured, and mutant algae strains with high oil yield were obtained through the three-index screening principle.

10. An algae-bacteria symbiotic system, characterized in that: The method according to claim 1 is used to strengthen the

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