CN114230015A - Method for removing nitrogen and phosphorus by utilizing constructed wetland microorganisms - Google Patents
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Abstract
The invention relates to the technical field of water environment treatment, and particularly discloses a method for removing nitrogen and phosphorus by utilizing constructed wetland microorganisms, which specifically comprises the steps of I, plant cultivation and benthos feeding; step two, water sample extraction and bacterial suspension preparation; step three, screening nitrogen and phosphorus degrading bacteria; step four, preparing a microbial inoculum; and step five, putting and applying. Aiming at the problem that the traditional constructed wetland has low efficiency of removing low-concentration nitrogen and phosphorus, the invention develops the constructed wetland plant-microorganism nitrogen and phosphorus reinforced removal technology, and the release application experiment shows that the release application is carried out in the experimental area of 10 hectares, compared with the same period in the last year, the removal improvement rate of TN reaches more than 30%, the removal improvement rate of TP exceeds 20%, and the removal effect of the constructed wetland on nitrogen and phosphorus is effectively improved.
Description
Technical Field
The invention relates to the technical field of water environment treatment, in particular to a method for removing nitrogen and phosphorus by utilizing constructed wetland microorganisms.
Background
A plurality of industrial parks are gathered in coastal cities in China, the industrial structure is heavier, and the problems of water environment, water ecology and water risk are prominent, and the typical expression of the method is as follows: the coastal industrial zone has high water impermeability and complex industrial types, so that the initial rainwater pollution degree is high; the tail water pollutant of the sewage treatment plant has complex components and remains toxic and harmful substances, wherein nitrogen and phosphorus at low concentration bottom are difficult to further purify deeply; meanwhile, the coastal beach wetland is lost or degraded, and the survival of waterfowls is threatened.
Aiming at the current situation of water environments of coastal industrial areas and artificial wetlands, the invention researches the technologies of low-concentration nitrogen and phosphorus artificial wetland enhanced removal, artificial wetland landscape construction and habitat restoration and plans to form an artificial wetland plant-microorganism nitrogen and phosphorus enhanced removal treatment technology.
Disclosure of Invention
The invention aims to provide a method for removing nitrogen and phosphorus by utilizing constructed wetland microorganisms, which realizes the purposes of reinforced removal of nitrogen and phosphorus of the constructed wetland and restoration of the ecological environment of the constructed wetland and achieves the comprehensive effects of cooperative removal of pollutants of the constructed wetland and ecological restoration.
In order to achieve the above object, the present invention provides a technical solution, which specifically comprises the following steps:
step S1: planting plants in the artificial wetland, and putting benthos;
step S2: adopt to (NH)4)2SO4And KNO3Separating and screening dominant degradation strains from the artificial wetland by using a nitrification culture medium and a denitrification culture medium which are nitrogen sources; taking a uniform mixing environment sampleThe product 10mL is placed in a conical flask containing 200mL inorganic salt liquid culture medium at 30 deg.C for 120r min-1Performing shake culture for 3-5 days to complete first enrichment, transferring 2mL of enrichment culture solution to an inorganic salt liquid culture medium with the same standard for enrichment culture for 2-3 times to prepare a bacterial suspension;
step S3: taking the enriched bacterial suspension, and setting the bacterial suspension to 10-1、10-2、10-3、10-4、10-5、10-6Respectively taking 0.1mL of bacterial suspension with 6 concentration gradients, respectively finishing coating on a nitrification solid culture medium and a denitrification solid culture medium, respectively selecting dominant strains with obvious morphological difference and the largest number of bacterial colonies after the bacterial colonies grow out, respectively scribing and separating on the corresponding solid culture media, placing the dominant strains in a constant temperature incubator at 30 ℃ for culture, selecting a single bacterial colony after the bacterial colony grows out again, continuously scribing on the corresponding solid culture medium, and repeating for 3-4 times to purify the bacterial strains; culturing at 10-30 ℃ to obtain pseudomonas aeruginosa, sword fungus adhesion, serratia marcescens and Pacific bacillus;
step S4: mixing the Jiancilia adherina, the Serratia marcescens and the Pacific bacillus screened in the step S3 according to a ratio of 2:1:1 to prepare a nitrogen-phosphorus composite degrading microbial inoculum; activating strains: inoculating the strain preserved in the freezing tube to a solid culture medium by a three-wire method to restore the activity of the strain; preparing a seed solution: selecting a single colony which grows well from the flat plate and inoculating the single colony in a liquid culture medium to prepare a fermentation seed solution; expanding culture: inoculating the seed liquid into a seed fermentation tank for secondary amplification culture; obtaining a microbial agent;
step S5: putting the microbial agent into the artificial wetland, adding a biological growth promoting agent, and putting again at intervals of 30-35 days, wherein the putting amount in the first three times is 180 kilograms per hectare; the amount put in the last three times is 100 kg/hectare.
The technical scheme aims at providing a method for removing nitrogen and phosphorus by utilizing constructed wetland microorganisms, which comprises the technologies of environmental sample collection and analysis, bacterial population diversity analysis, bacterial population composition and influence factor analysis, nitrogen degrading bacteria screening, phosphorus degrading bacteria screening, composite microbial inoculum preparation, composite degrading microbial inoculum putting in the constructed wetland, on-site degradation effect analysis and the like, aiming at the problem that the traditional constructed wetland has low efficiency of removing low-concentration nitrogen and phosphorus, the microorganism nitrogen and phosphorus enhanced removal technology is developed, namely, denitrification and phosphorus accumulating bacteria are screened from the root system of aquatic plants of the constructed wetland, the nitrogen and phosphorus degrading microbial inoculum is prepared by integrating a plurality of technologies of a high-flux fermentation technology for degrading microorganisms, an enzyme-fermentation coupling high-efficiency conversion technology and the like, the biological enhanced system is successfully constructed in the constructed wetland system and is scientifically matched with a biological growth promoter to improve the pollutant removal capability of the microorganisms at the roots of the plants, promoting the synergistic removal effect of the plants and the microorganisms of the wetland system on nitrogen and phosphorus.
Furthermore, in step S1, in the artificial wetland, emergent aquatic plants, such as reed, cattail, rush, tatarian aster root, suaeda salsa, swertia, setaria viridis, endive, burclover, kochia scoparia, droughhaired umbrella herb, vetiver and canna, which can grow rapidly and are exuberant, are planted in the artificial wetland; and putting benthonic animals such as Japanese drum shrimps, crisp shell clams, small walnut clams, villous crayfish and the like.
Further, the inorganic salt liquid culture medium in the step S2 is KNO3 1.0g、K2HPO4 0.5g、KH2PO40.5g、MgSO4·7H2O 0.5g、NaCl 1.0g、CaCl2 0.1g、FeCl30.02g and 0.05g of yeast extract, and the volume is determined to be 1000mL after dissolution, and the pH value is 7.0-7.2.
Further, the nitrifying medium in the step S2 is (NH)4)2SO40.5g, 2.17g of sodium succinate and 50mL of Vickers salt solution, adding water for dissolving, and supplementing distilled water to 1000 mL; vickers salt solution K2HPO4 5.0g、MgSO4·7H2O 2.5g、NaCl 2.5g、FeSO4·7H2O 0.05g、MnSO4·4H20.05g of O, adding water after dissolution to fix the volume to 1000mL of culture medium; the nitrifying solid medium in the step S2 is obtained by adding 2% agar into the nitrifying medium and sterilizing for 30min at the temperature of 118 ℃.
Further, the denitrification culture medium in the step S2 is 3g of beef extract and peptone5g、KNO31g of culture medium with 1000mL of distilled water and pH value of 7.0-7.6; the denitrification solid culture medium in the step S2 is obtained by adding 2% agar into the denitrification culture medium and sterilizing for 30min at 118 ℃.
Further, the environmental sample in step S2 is first filtered with a filter membrane smaller than 8 μm after collection to remove the suspended matter with larger particle size in the environmental sample.
Further, the culture temperature in the step S3 is 20-25 ℃.
Further, in the step S4, the strain is activated and cultured in a constant temperature incubator at the temperature of 35 ℃ for 18-24 hours.
Further, the culture conditions for preparing the seed solution in the step S4 are as follows: the culture temperature is 35 +/-1 ℃, the pressure is 0.1-0.12 MPa, and the seed culture time is 16-18 h.
Compared with the prior art, the invention has the overall technical effects that:
the technology of the invention is applied to screening three indigenous high-efficiency nitrogen and phosphorus degradation strains (Jianjun adhesion bacteria, Serratia marcescens and Pacific bacillus) on the root system of the aquatic plant (mainly reed) of the constructed wetland, the three strains are mixed according to the proportion of 2:1:1 to prepare a composite degradation microbial inoculum, a biological growth promoter is added organically, the composite degradation microbial inoculum is sprayed on the areas such as a wetland water inlet, the constructed wetland, a reed planting area and the like, compared with TN (total nitrogen) and TP (total phosphorus) monitoring data of the wetland in the past year, the removal effect is evaluated by adopting the monitoring data of a third-party evaluation unit, and the result shows that: compared with the same period of 2019 in 2020, the removal improvement rate of TN is more than 30%, the removal improvement rate of TP is more than 20%, and the removal effect of nitrogen and phosphorus of the constructed wetland is effectively improved. The application of the technology of the invention shows that the water quality purification effect and the habitat restoration effect are both obviously improved.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a process flow diagram of an embodiment of the invention;
FIG. 2 is a graph showing growth curves of respective strains at different temperatures;
FIG. 3 shows the degradation rate of TN by different strains under different temperature conditions;
FIG. 4 shows the TP degradation rate of different strains under different temperature conditions;
FIG. 5 shows the degradation rates of TN and TP by the composite degrading strain under different temperature conditions.
Wherein: a-pseudomonas aeruginosa, B-swordlike bacillus sticktight, C-serratia marcescens and D-Paenibacillus pacificus.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In the description of the present invention, it is to be understood that the terms "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be construed as limiting the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the present invention, unless otherwise expressly stated or limited, "above" or "below" a first feature means that the first and second features are in direct contact, or that the first and second features are not in direct contact but are in contact with each other via another feature therebetween. Also, the first feature being "on," "above" and "over" the second feature includes the first feature being directly on and obliquely above the second feature, or merely indicating that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature includes the first feature being directly under and obliquely below the second feature, or simply meaning that the first feature is at a lesser elevation than the second feature.
Example (b):
a method for removing nitrogen and phosphorus by utilizing constructed wetland microorganisms specifically comprises the following steps:
step S1: planting reed, typha orientalis, rush, radix asteris, suaeda salsa, swertia pseudochinensis, setaria viridis, endive, burclover, broom cypress, droughhaired holly herb, vetch and other root systems in the artificial wetland, wherein the root systems can grow rapidly and are exuberant emergent aquatic plants; and putting benthonic animals such as Japanese drum shrimps, crisp shell clams, small walnut clams, villous crayfish and the like;
step S2: taking 10mL of a uniformly mixed environment sample from the plant root system sediment or water body of the artificial wetland, placing the uniformly mixed environment sample in a conical flask containing 200mL of inorganic salt liquid culture medium, carrying out shaking culture at 30 ℃ for 3-5 d to complete first enrichment, taking 2mL of the enriched culture solution, transferring the enriched culture solution to the inorganic salt liquid culture medium with the same standard, carrying out enrichment culture for 2-3 times, and preparing to obtain a bacterial suspension;
step S3: collecting the enriched bacterial liquid, and setting to 10-1、10-2、10-3、10-4、10-5、10-6Respectively taking 0.1mL of bacterial suspension with 6 concentration gradients, respectively finishing coating on a nitrification solid culture medium and a denitrification solid culture medium, selecting a flat plate with proper concentration to respectively select dominant strains with obvious morphological difference and the largest number of bacterial colonies and the largest number of the dominant strains on the two culture media after bacterial colonies grow out, respectively scribing and separating on the corresponding solid culture media, placing the dominant strains in a constant-temperature incubator at 30 ℃ for culture, picking a single bacterial colony after the bacterial colony grows out again, continuously scribing on the corresponding solid culture medium, and repeating for 3-4 times to purify the bacterial strains; obtaining 4 strains which are respectively named as A, B, C, D, wherein A and B are obtained by separation on a nitrification culture medium, and C and D are obtained by separation on a denitrification culture medium; through identification, the A bacterial strain is Pseudomonas aeruginosa (Pseudomonas aeruginosa), the bacterium grows well on a common culture medium and is an obligate aerobic bacterium, and nitrate can be used as a hydrogen acceptor to grow under an anaerobic condition; the B strain is Ensifer adhaerens (Ensifer adhaerens), and the strain has certain removal effect on nitrogen ammonia, TP, Chemical Oxygen Demand (COD) in sewage, Cu, Pb, As and the like in the sewage; the strain C is Serratia marcescens (Serratia marcescens), has high phosphorus accumulation capacity and plays an important role in removing phosphorus; the strain D is Pacific Bacillus (Bacillus pacificus), which can degrade COD in water treatment and has obvious assimilation denitrification effect;
step S4: mixing the strain D, the strain C and the strain D screened in the step S3 according to a ratio of 2:1:1 to prepare a nitrogen-phosphorus composite degrading microbial inoculum; activating strains: inoculating the strain preserved in the freezing tube to a solid culture medium by a three-wire method to restore the activity of the strain; preparing a seed solution: selecting a single colony which grows well from the flat plate and inoculating the single colony in a liquid culture medium to prepare a fermentation seed solution; expanding culture: inoculating the seed liquid into a seed fermentation tank for secondary amplification culture; obtaining a microbial agent;
step S5: putting the microbial agent into the artificial wetland, adding a biological growth promoting agent, and putting again at intervals of 30-35 days, wherein the putting amount in the first three times is 180 kilograms per hectare; the later three times of putting is 100 kilograms per hectare; wherein, respectively weighing 25g of yeast extract, 3.6g of aspartic acid, 14.4g of tryptophan, 0.5g of vitamin H1g, 0.45g of sodium chloride, 0.45g of 6-benzyl purine and other trace elements, mixing and dissolving in 200mL of distilled water, filtering, removing residues, and obtaining filtrate, namely the prepared biological growth promoter, wherein in order to ensure that the biological growth promoter can play a normal role in the whole implementation stage, the biological growth promoter is placed in a refrigerator at 4 ℃ for storage for later use, and is mixed with the complex microbial inoculum for sample injection each time during addition; when the microbial inoculum is put into the water distribution channel of the wetland, the regulating pond and the subsurface flow wetland, the microbial inoculum is directly and uniformly poured into water at different positions, and other areas adopt a suspension high-pressure spraying device to spray the microbial inoculum on the reed root system area, so that the microbial inoculum can be colonized and colonized as soon as possible.
In order to make the aspects and effects of the embodiments more directly and more comprehensible, the present embodiment is described in further detail below with reference to the accompanying drawings.
FIG. 1 is a process flow diagram of an embodiment of the present invention, described in detail in the embodiment.
FIG. 2 is a growth curve of each strain at different temperatures, specifically:
according to the growth curve of the strain within 96h at the temperature of 5 ℃, 13 ℃, 20, 27 and 34 ℃, 4 bacteria can grow exponentially within 48h, but the proliferation rates are different to a certain extent, and the whole strain is obviously increased along with time, which indicates that the strain grows better. The wetland influent raw water can meet the nutrition requirement of the strain growth within 96 hours of experiment time.
Under the temperature conditions of 5, 13, 20, 27 and 34 ℃, the OD600 value of the pseudomonas aeruginosa A basically grows exponentially within 48h, which shows that the bacteria are in logarithmic growth phase, the OD600 value still grows within 48-96 h, but the increment is small, the bacteria enter stable growth phase, which shows that nutrient substances in the culture environment can meet the requirement of the growth of the bacteria, and the bacteria can grow well within 48 h. In contrast, under the condition of 5 ℃, the OD600 value of the bacteria shows a continuous rising trend within 96h, but the overall increment is small and is only 0.299, which indicates that under the environment of 5 ℃, the activity of the bacteria is relatively low, the bacteria grow slowly, but under the condition of sufficient nutrient substances, the bacteria can still adapt to the environment to survive.
The Exoecaria californica B can grow well at the temperature of 13 ℃, 20 ℃, 27 ℃ and 34 ℃, wherein the Exoecaria californica B has more advantages in growth at 20 ℃, 27 ℃ and 34 ℃, the OD600 value can reach 1.91 at most, the maximum value of the OD600 value can also reach more than 1.35 at 13 ℃, and the Exoecaria californica B grows well at 5 ℃, which indicates that the temperature application range of the strain is wider.
The serratia marcescens C can grow well under the temperature conditions of 13 ℃, 20 ℃, 27 ℃ and 34 ℃, wherein the growth at 20 ℃ and 27 ℃ is more advantageous, the OD600 value can reach 1.3 at most, and the OD600 value can also reach more than 1.0 under the conditions of 13 ℃ and 34 ℃, which shows that the temperature adaptation range of the strain is wider, the OD600 value increment is higher and is 0.336 at 5 ℃, and the growth curve trends show regular logarithmic growth period and stable growth period under other temperature conditions.
The Pacific bacillus D has good growth vigor, the OD600 value can reach 1.4 when the Pacific bacillus D is cultured for 48 hours at the temperature of 13 ℃, the temperature influence difference of the Pacific bacillus D to the Pacific bacillus D is relatively unobvious at the temperature of 13 ℃, 20 ℃, 27 ℃ and 34 ℃, the OD600 value increment at the temperature of 5 ℃ is also high and is 0.382, previous researches show that the Pacific bacillus D can generate spores resisting adverse environmental conditions, sporulation is not inhibited by oxygen, the Pacific bacillus D is high-temperature resistant, can tolerate the high temperature of 60 ℃ for a long time, can survive for 20min at the temperature of 120 ℃, is acid-base resistant, and can keep activity in a gastric acid environment.
In summary, the OD600 values of the 4 strains basically increase exponentially within 48 hours at different temperatures, the bacteria enter a stable growth period within 48-96 hours, which shows that nutrients in a culture environment can meet the requirements of the bacteria for growth, the bacteria can proliferate faster within 48 hours, the strains have certain difference as soon as possible, and the bacteria grow relatively slowly at 5 ℃, but the results show that the growth temperatures of the 4 strains are relatively wide in application range, and the strains can grow better in spring, summer and autumn and winter of the constructed wetland.
FIG. 3 shows the degradation rate of TN by different strains under different temperature conditions. The degradation of NH4-N in wetland influent water by 4 strains at different temperatures is shown in figure 3, and the degradation rates of raw water NH4-N by pseudomonas aeruginosa A are 3.9%, 2.8%, 5.9%, 8.8% and 10.9% respectively; the degradation rates of the Ensifer adhesivelis B are respectively 18.5%, 40.4%, 60.9%, 80.5% and 83.9%; the degradation rates of the serratia marcescens C are respectively 1.1%, 0.6%, 2.4%, 6.3% and 4.5%; the degradation rates of Pacific bacillus D were 20.2%, 29.8%, 72.8%, 73.0%, 74.7%, respectively. Similarly, E.viscosus and Bacillus exhibit higher degradation efficiency for NH4-N, giving the highest degradation rates at 34 ℃ of 83.8% and 74.8%, respectively, whereas Pseudomonas aeruginosa and Serratia at different temperatures have lower degradation rates ranging from 2.9% to 10.9% and from 0.6% to 6.3%, respectively, with optimal degradation occurring at 27 ℃ and 34 ℃, respectively.
FIG. 4 shows the TP degradation rate of different strains under different temperature conditions. The results of the degradation efficiency study of two strains on TP in wetland raw water at different temperatures (5, 13, 20, 27 and 34 ℃) are shown in FIG. 4. In the whole, the two strains have certain phosphorus accumulation capacity within 48 hours. 5. Under the conditions of 13, 20, 27 and 34 ℃, the average degradation rates of the sword iron bacterium B to TP are respectively 13.6%, 19.9%, 36.6%, 34.5% and 54.3%, and the degradation rates of the serratia marcescens C to TP are respectively 25.4%, 41.3%, 63.3%, 63.9% and 65.9%. The degradation rate is increased along with the increase of the temperature, and the degradation efficiency of the strain C is higher than that of the strain B.
FIG. 5 shows the degradation rates of TN and TP by the composite degrading strain under different temperature conditions. Research on the degradation efficiency of the composite degradation microbial inoculum on nitrogen and phosphorus in wetland inlet raw water at different temperatures is carried out in a laboratory, and the result is shown in fig. 5. In an overall view, the composite degradation microbial inoculum has certain degradation to TN and TP under different temperature conditions, under the conditions of 5, 13, 20, 27 and 34 ℃, the average degradation rate to TN within 48h is 17.0%, 29.3%, 50.9%, 61.6% and 62.4% respectively, and the average degradation rate to TP is 8.3%, 24.4%, 44.8%, 42.4% and 45.4% respectively. The degradation rate is increased along with the increase of the temperature, and the degradation rate of TN is higher than that of TP at the same temperature. The degradation rate of the raw water TN can reach 62.4% under the condition of proper temperature, the dominant degradation capability is shown, and meanwhile, the mixed bacteria also show good degradation efficiency on the raw water TP, so that the composite microbial inoculum can be applied to large-scale treatment of nitrogen and phosphorus sewage, has wide application prospect, and provides new technical reference and reference for purification and restoration of low-nitrogen and phosphorus polluted water.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (9)
1. A method for removing nitrogen and phosphorus by utilizing constructed wetland microorganisms is characterized by comprising the following steps:
s1, planting plants in the artificial wetland, and putting benthos;
s2, taking 10mL of the mixed environment sample from the plant root system sediment or the water body of the artificial wetland, uniformly mixing the mixed environment sample with 200mL of the conical flask containing the inorganic salt liquid culture medium, and carrying out the treatment at the temperature of 30 ℃ for 120 r.min-1Performing shake culture for 3-5 days to complete first enrichment, transferring 2mL of enrichment culture solution to an inorganic salt liquid culture medium with the same standard for enrichment culture for 2-3 times to prepare a bacterial suspension;
s3, adopting (NH)4)2SO4And KNO3Separating and screening dominant degradation strains from the bacterial suspension by using a nitrification culture medium and a denitrification culture medium which are nitrogen sources; collecting the enrichedThe bacterial suspension is set to 10-1、10-2、10-3、10-4、10-5、10-6Respectively taking 0.1mL of bacterial suspension with 6 concentration gradients, respectively finishing coating on a nitrification solid culture medium and a denitrification solid culture medium, respectively selecting dominant strains with obvious morphological difference and the largest number of bacterial colonies after the bacterial colonies grow out, respectively scribing and separating on the corresponding solid culture media, placing the dominant strains in a constant temperature incubator at 30 ℃ for culture, selecting a single bacterial colony after the bacterial colony grows out again, continuously scribing on the corresponding solid culture medium, and repeating for 3-4 times to purify the bacterial strains; culturing at 10-30 ℃ to obtain pseudomonas aeruginosa (A), swordlike bacillus adhesion (B), serratia marcescens (C) and Pacific bacillus (D);
s4, mixing the Jianbaena adhensium (B), the Serratia marcescens (C) and the Pacific bacillus (D) screened in the step S3 according to a ratio of 2:1:1 to prepare a nitrogen-phosphorus composite degrading microbial inoculum; activating strains: inoculating the strain preserved in the freezing tube to a solid culture medium by a three-wire method to restore the activity of the strain; preparing a seed solution: selecting a single colony which grows well from the flat plate and inoculating the single colony in a liquid culture medium to prepare a fermentation seed solution; expanding culture: inoculating the seed liquid into a seed fermentation tank for secondary amplification culture; obtaining a microbial agent;
s5, putting the microbial agent into the artificial wetland, adding a biological growth promoting agent, and putting again at intervals of 30-35 days, wherein the putting amount of the first three times is 180 kilograms per hectare; the amount put in the last three times is 100 kg/hectare.
2. The method for removing nitrogen and phosphorus by using constructed wetland microorganisms as claimed in claim 1, wherein the plant in step S1 is one or more of reed, cattail, radix asteris, suaeda salsa, swertia pseudochinensis, setaria japonica, endive, burclover and kochia scoparia, and the benthic organism is one or more of japanese drum shrimp, crunchy clam, walnut clam and crab with villous foot.
3. The method for removing nitrogen and phosphorus by using constructed wetland microorganisms as claimed in claim 1,the inorganic salt liquid culture medium in the step S2 is KNO3 1.0g、K2HPO4 0.5g、KH2PO4 0.5g、MgSO4·7H2O 0.5g、NaCl 1.0g、CaCl2 0.1g、FeCl30.02g and 0.05g of yeast extract, and the volume is determined to be 1000mL after dissolution, and the pH value is 7.0-7.2.
4. The method for removing nitrogen and phosphorus by using constructed wetland microorganisms as claimed in claim 1, wherein the nitrification medium in step S2 is (NH)4)2SO40.5g, 2.17g of sodium succinate and 50mL of Vickers salt solution, adding water for dissolving, and supplementing distilled water to 1000 mL; vickers salt solution K2HPO4 5.0g、MgSO4·7H2O 2.5g、NaCl 2.5g、FeSO4·7H2O 0.05g、MnSO4·4H20.05g of O, adding water after dissolution to fix the volume to 1000mL of culture medium; the nitrifying solid medium in the step S2 is obtained by adding 2% agar into the nitrifying medium and sterilizing for 30min at the temperature of 118 ℃.
5. The method for removing nitrogen and phosphorus by using constructed wetland microorganisms as claimed in claim 1, wherein the denitrification medium in the step S2 is beef extract 3g, peptone 5g, KNO31g of culture medium with 1000mL of distilled water and pH value of 7.0-7.6; the denitrification solid culture medium in the step S2 is obtained by adding 2% agar into the denitrification culture medium and sterilizing for 30min at 118 ℃.
6. The method for removing nitrogen and phosphorus by using constructed wetland microorganisms as claimed in any one of claims 1 to 5, wherein the environmental sample in the step S2 is collected and filtered by a filter membrane smaller than 8 μm to remove suspended matters with larger particle size in the environmental sample.
7. The method for removing nitrogen and phosphorus by using constructed wetland microorganisms as claimed in claim 6, wherein the culture temperature in the step S3 is 20-25 ℃.
8. The method for removing nitrogen and phosphorus by using constructed wetland microorganisms as claimed in claim 6, wherein the strain activation in step S4 is carried out in a constant temperature incubator at 35 ℃ for 18-24 h.
9. The method for removing nitrogen and phosphorus by using constructed wetland microorganisms as claimed in claim 6, wherein the culture conditions for preparing the seed solution in the step S4 are as follows: the culture temperature is 35 +/-1 ℃, the pressure is 0.1-0.12 MPa, and the seed culture time is 16-18 h.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480207A (en) * | 2022-02-22 | 2022-05-13 | 青岛蔚蓝赛德生物科技有限公司 | Pacific bacillus and application thereof in degradation of sulfide in sewage wastewater |
| CN116254192A (en) * | 2022-11-29 | 2023-06-13 | 云南大学 | Pacific bacillus rice strain for producing siderophore and application thereof |
| CN116904349A (en) * | 2023-06-15 | 2023-10-20 | 中国科学院上海高等研究院 | Adhesive sword bacteria with aerobic denitrification capability and application thereof |
-
2021
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480207A (en) * | 2022-02-22 | 2022-05-13 | 青岛蔚蓝赛德生物科技有限公司 | Pacific bacillus and application thereof in degradation of sulfide in sewage wastewater |
| CN114480207B (en) * | 2022-02-22 | 2023-09-22 | 青岛蔚蓝赛德生物科技有限公司 | Pacific bacillus and application thereof in degrading sulfides in sewage and wastewater |
| CN116254192A (en) * | 2022-11-29 | 2023-06-13 | 云南大学 | Pacific bacillus rice strain for producing siderophore and application thereof |
| CN116254192B (en) * | 2022-11-29 | 2024-02-23 | 云南大学 | Pacific bacillus rice strain for producing siderophore and application thereof |
| CN116904349A (en) * | 2023-06-15 | 2023-10-20 | 中国科学院上海高等研究院 | Adhesive sword bacteria with aerobic denitrification capability and application thereof |
| CN116904349B (en) * | 2023-06-15 | 2024-03-22 | 中国科学院上海高等研究院 | Adhesive sword bacteria with aerobic denitrification capability and application thereof |
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